Prostate-associated antigens and vaccine-based immunotherapy regimens

ABSTRACT

The present disclosure provides (a) isolated immunogenic PAA polypeptides; (b) isolated nucleic acid molecules encoding immunogenic PAA polypeptides; (c) vaccine compositions comprising an immunogenic PAA polypeptide or an isolated nucleic acid molecule encoding an immunogenic PAA polypeptide; (d) methods relating to uses of the polypeptides, nucleic acid molecules, and compositions; and (e) vaccine-based immunotherapy regimens which involve co-administration of a vaccine in combination with an immune-suppressive-cell inhibitor and an immune-effector-cell enhancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of application Ser. No. 13/875,162 filedon May 1, 2013, now allowed, which claims benefit of U.S. ProvisionalApplication No. 61/642,844 filed on May 4, 2012. Both application Ser.No. 13/875,162 and U.S. Provisional Application No. 61/642,844 areincorporated herein by reference in their entirety.

REFERENCE TO SEQUENCE LISTING

This application is being filed along with a sequence listing inelectronic format. The sequence listing is provided as a file in .txtformat entitled “PC71854A SEQ LISTING_ST25.TXT”, created on Apr. 4, 2013and having a size of 257 KB. The sequence listing contained in the .txtfile is part of the specification and is herein incorporated byreference in its entity.

FIELD OF THE INVENTION

The present invention relates generally to immunotherapy andspecifically to vaccines and methods for treating or preventingneoplastic disorders.

BACKGROUND OF THE INVENTION

Cancer is a leading cause of mortality worldwide. Traditional regimensof cancer management have been successful in the management of aselective group of circulating and solid cancers. However, many tumorsare resistant to traditional approaches. In recent years, immunotherapyfor the treatment of cancers has been explored, which involves thegeneration of an active systemic tumor-specific immune response of hostorigin by administering a vaccine composition at a site distant from thetumor. Various types of vaccines have been proposed, including thosecontaining isolated tumor-associated antigens.

Prostate cancer is the second most commonly diagnosed cancer and thefourth leading cause of cancer-related death in men in the developedcountries worldwide. Various prostate-associated antigens (PAA), such asprostate-specific antigen (PSA), prostate-specific membrane antigen(PSMA), and prostate stem cell antigen (PSCA) have been shown to beoverexpressed by prostate cancer cells as compared to normalcounterparts. These antigens, therefore, represent possible targets forinducing specific immune responses against cancers expressing theantigens via the use of vaccine-based immunotherapy. (see e.g. Marrari,A., M. lero, et al. (2007). “Vaccination therapy in prostate cancer.”Cancer Immunol Immunother 56(4): 429-45.)

PSCA is a 123-amino acid membrane protein. The amino acid sequence ofthe full length human PSCA consists of amino acids 4-123 of SEQ IDNO:21. PSCA has high tissue specificity and is expressed on more than85% of prostate cancer specimens, with expression levels increasing withhigher Gleason scores and androgen independence. It is expressed in80-100% of bone metastasis of prostate cancer patients.

PSA is a kallikrein-like serine protease that is produced exclusively bythe columnar epithelial cells lining the acini and ducts of the prostategland. PSA mRNA is translated as an inactive 261-amino acid preproPSAprecursor. PreproPSA has 24 additional residues that constitute thepre-region (the signal polypeptide) and the propolypeptide. Release ofthe propolypeptide results in the 237-amino acid, mature extracellularform, which is enzymatically active. The amino acid sequence of thehuman full length PSA is provided in SEQ ID NO: 15. PSA isorgan-specific and, as a result, it is produced by the epithelial cellsof benign prostatic hyperplastic (BPH) tissue, primary prostate cancertissue, and metastatic prostate cancer tissue.

PSMA, also known as Folate hydrolase 1 (FOLH1), is composed of 750 aminoacids. The amino acid sequence of the human full length PSMA is providedin SEQ ID NO:1. PSMA includes a cytoplasmic domain (amino acids 1-19), atransmembrane domain (amino acids 20-43), and an extracellular domain(amino acids 44-750). PSMA is a type II dimeric transmembrane proteinexpressed on the surface of prostate cancer cells and on neovasculature.It is also expressed on normal prostate cells, brain, salivary gland andbiliary tree. However, in prostate cancer cells it was found to beexpressed at 1000-fold higher levels than normal tissues. It isabundantly expressed on neovasculature of a variety of other solidtumors such as colon, breast, liver, bladder, pancreas, lung, renalcancers as well as melanoma and sarcomas. Thus, PSMA is considered atarget not only specific for prostate cancer cells but also apan-carcinoma target for other cancers. The expression of PSMA appearsto be a universal feature of prostate carcinomas and its increasedexpression correlates with tumor aggressiveness. PSMA expression ishighest in high-grade tumors, metastatic lesions andandrogen-independent disease.

While a large number of tumor-associated antigens have been identifiedand many of these antigens have been explored as protein-based orDNA-based vaccines for the treatment or prevention of cancers, mostclinical trials so far have failed to produce a therapeutic product. Oneof the challenges in developing cancer vaccines resides in the fact thatthe cancer antigens are usually self-derived and, therefore, poorlyimmunogenic because the immune system is self-regulated not to recognizeself-proteins. Accordingly, a need exists for a method to enhance theimmunogenicity or therapeutic effect of cancer vaccines.

Numerous approaches have been explored for enhancing the immunogenicityor enhancing anti-tumor efficacy of cancer vaccines. One of suchapproach involves the use of various immune modulators, such as TLRagonists, TNFR agonists, CTLA-4 inhibitors, and protein kinaseinhibitors.

Toll-like receptors (TLRs) are type 1 membrane receptors that areexpressed on hematopoietic and non-hematopoietic cells. At least 11members have been identified in the TLR family. These receptors arecharacterized by their capacity to recognize pathogen-associatedmolecular patterns (PAMP) expressed by pathogenic organisms. It has beenfound that triggering of TLR elicits profound inflammatory responsesthrough enhanced cytokine production, chemokine receptor expression(CCR2, CCR5 and CCR7), and costimulatory molecule expression. As such,these receptors in the innate immune systems exert control over thepolarity of the ensuing acquired immune response. Among the TLRs, TLR9has been extensively investigated for its functions in immune responses.Stimulation of the TLR9 receptor directs antigen-presenting cells (APCs)towards priming potent, T_(H)1-dominated T-cell responses, by increasingthe production of pro-inflammatory cytokines and the presentation ofco-stimulatory molecules to T cells. CpG oligonucleotides, ligands forTLR9, were found to be a class of potent immunostimulatory factors. CpGtherapy has been tested against a wide variety of tumor models in mice,and has consistently been shown to promote tumor inhibition orregression.

Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) is a member of theimmunoglobulin superfamily and is expressed on the surface of Helper Tcells. CTLA-4 is a negative regulator of CD28 dependent T cellactivation, and acts as an inhibitory checkpoint for the adaptive immuneresponse. Similar to the T-cell costimulatory protein CD28, CTLA-4 bindsto CD80 and CD86 on antigen-presenting cells. CTLA-4 transmits aninhibitory signal to T cells, whereas CD28 transmits a stimulatorysignal. Human antibodies against human CTLA-4 have been described asimmunostimulation modulators in a number of disease conditions, such astreating or preventing viral and bacterial infection and for treatingcancer (WO 01/14424 and WO 00/37504). Various preclinical studies haveshown that CTLA-4 blockade by monoclonal antibodies enhances the hostimmune response against immunogenic tumors, and can even rejectestablished tumors. Two fully human anti-human CTLA-4 monoclonalantibodies (mAbs), ipilimumab (MDX-010) and Tremelimumab (also known asCP-675206), have been investigated in clinical trials in the treatmentof various types of solid tumors.

The tumor necrosis factor (TNF) superfamily is a group of cytokines thatengage specific cognate cell surface receptors, the TNF receptor (TNFR)superfamily. Members of the tumor necrosis factor superfamily actthrough ligand-mediated trimerization, causing recruitment of severalintracellular adaptors to activate multiple signal transductionpathways, such as apoptosis, NF-kB pathway, JNK pathway, as well asimmune and inflammatory responses. Examples of the TNF Superfamilyinclude CD40 ligands, OX40 ligands, 4-1BB ligands, CD27, CD30 ligand(CD153), TNF-alpha, TNF-beta, RANK ligands, LT-alpha, LT-beta, GITRligands, and LIGHT. The TNFR Superfamily includes, for example, CD40,OX40, 4-1BB, CD70 (CD27 ligand), CD30, TNFR2, RANK, LT-beta R, HVEM,GITR, TROY, and RELT. CD40 is found on the surface of B lymphocytes,dendritic cells, follicular dendritic cells, hematopoietic progenitorcells, epithelial cells, and carcinomas. CD40 binds to a ligand(CD40-L), which is a glycoprotein and expressed on activated T cells,mostly CD4+ but also some CD8+ as well as basophils/mast cells. Becauseof the role of CD40 in innate and adaptive immune responses, CD40agonists, including various CD40 agonistic antibodies, such as the fullyhuman agonist CD40 monoclonal antibody CP870893, have been explored forusage as vaccine adjuvants and in therapies.

Protein kinases are a family of enzymes that catalyze thephosphorylation of specific residues in proteins. Protein kinases arekey elements in signal transduction pathways responsible for transducingextracellular signals, including the action of cytokines on theirreceptors, to the nuclei, triggering various biological events. The manyroles of protein kinases in normal cell physiology include cell cyclecontrol and cell growth, differentiation, apoptosis, cell mobility andmitogenesis. Kinases such as c-Src, c-Abl, mitogen activated protein(MAP) kinase, phosphotidylinositol-3-kinase (PI3K) AKT, and theepidermal growth factor (EGF) receptor are commonly activated in cancercells, and are known to contribute to tumorigenesis. Logically, a numberof kinase inhibitors are currently being developed for anti-cancertherapy, in particular tyrosine kinase inhibitors (TKIs):cyclin-dependent kinase inhibitors, aurora kinase inhibitors, cell cyclecheckpoint inhibitors, epidermal growth factor receptor (EGFR)inhibitors, FMS-like tyrosine kinase inhibitors, platelet-derived growthfactor receptor (PDGFR) inhibitors, kinase insert domain inhibitors,inhibitors targeting the PI3K/Akt/mTOR pathway, inhibitors targeting theRas-Raf-MEK-ERK (ERK) pathway, vascular endothelial growth factorreceptor (VEGFR) kinase inhibitors, c-kit inhibitors andserine/threonine kinase inhibitors. A number of kinase inhibitors havebeen investigated in clinical investigation for use in anti-cancertherapies, which includes, for example, MK0457, VX-680, ZD6474, MLN8054,AZD2171, SNS-032, PTK787/ZK222584, Sorafenib (BAY43-9006), SU5416,SU6668 AMG706, Zactima (ZD6474), MP-412, Dasatinib, CEP-701,(Lestaurtinib), XL647, XL999, Tykerb, (Lapatinib), MLN518, (formerlyknown as CT53518), PKC412, ST1571, AMN107, AEE 788, OSI-930, OSI-817,Sunitinib malate (Sutent; SU11248), Vatalanib (PTK787/ZK 222584),SNS-032, SNS-314 and Axitinib (AG-013736). Gefitinib and Erlotinib aretwo orally available EGFR-TKIs.

The immune modulators that have been explored are typically administeredsystemically to the patients, for example, by oral administration,intravenous injection or infusion, or intramuscular injection. One majorfactor that limits the effective use of some of the immune modulators istoxicity caused by high systemic exposure to the administered agents.For example, with respect to CD40 agonists, it has been reported that0.3 mg/kg is the maximum tolerated dose for an exemplified agonisticCD40 antibody and that higher doses may elicit side effects includingvenous thromboembolism, grade 3 headache, cytokine release resulting intoxic effects such as chills and the like, and transient liver toxicity.(Vanderheide et al., J Clin. Oncol. 25(7): 876-8833 (March 2007). In aclinical trial to investigate combinations of intravenous Tremelimumab(an anti-CTLA-4 antibody) plus oral sunitinib in patients withmetastatic renal cell carcinoma, rapid onset of renal failure wasobserved and, as a result, further investigation of Tremelimumab atdoses higher than 6 mg/kg plus sunitinib at 37.5 mg daily was notrecommended. See: Brian I. Rini et al.: Phase 1 Dose-Escalation Trial ofTremelimumab Plus Sunitinib in Patients With Metastatic Renal CellCarcinoma. Cancer 117(4)158-767 (2011)]. Therefore, there is a need forvaccine-based immunotherapy regimens where the immune modulators areadministered at effective doses which do not elicit severe adverse sideeffects such as liver toxicity or renal failure.

SUMMARY OF THE INVENTION

In some aspects, the present disclosure provides isolated immunogenicPSMA polypeptides and immunogenic PSA polypeptides, which are useful,for example, for eliciting an immune response in vivo (e.g. in ananimal, including humans) or in vitro, generating antibodies, or for useas a component in vaccines for treating cancers, including prostatecancer. In one aspect, the present disclosure provides isolatedimmunogenic PSMA polypeptides which have at least 90% identity to aminoacids 15-750 of the human PSMA of SEQ ID NO:1 and comprise the aminoacids of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of theconserved T cell epitopes of the human PSMA at corresponding positions.

In other aspects, the present disclosure provides nucleic acid moleculesthat encode immunogenic PAA polypeptides. In some embodiments, thepresent disclosure provides isolated nucleic acid molecules, ordegenerate variants thereof, which comprise a nucleotide sequenceencoding an immunogenic PSMA polypeptide, or a functional variant ofsaid polypeptide, provided by the present disclosure.

In some other aspects, the present disclosure provides multi-antigennucleic acid constructs that each encode two or more immunogenic PAApolypeptides.

The disclosure also provides vectors containing one or more nucleic acidmolecules of the invention. The vectors are useful for cloning orexpressing the immunogenic PAA polypeptides encoded by the nucleic acidmolecules, or for delivering the nucleic acid molecules in acomposition, such as a vaccine, to a host cell or to a host animal, suchas a human.

In some further aspects, the present disclosure provides compositionscomprising one or more immunogenic PAA polypeptides, isolated nucleicacid molecules encoding immunogenic PAA polypeptides, or vectors orplasmids containing nucleic acid molecules encoding immunogenic PAApolypeptides. In some embodiments, the composition is an immunogeniccomposition useful for eliciting an immune response against a PAA in amammal, such as a mouse, dog, monkey, or human. In some embodiments, thecomposition is a vaccine composition useful for immunization of amammal, such as a human, for inhibiting abnormal cell proliferation, forproviding protection against the development of cancer (used as aprophylactic), or for treatment of disorders (used as a therapeutic)associated with PAA over-expression, such as cancer, particularlyprostate cancer.

In still other aspects, the present disclosure provides methods of usingthe immunogenic PAA polypeptides, isolated nucleic acid molecules, andcompositions comprising an immunogenic PAA polypeptide or isolatednucleic acid molecules described herein above. In some embodiments, thepresent disclosure provides a method of eliciting an immune responseagainst a PAA in a mammal, particularly a human, comprisingadministering to the mammal an effective amount of a polypeptideprovided by the invention that is immunogenic against the target PAA, aneffective amount of an isolated nucleic acid molecule encoding such animmunogenic polypeptide, or a composition comprising such an immunogenicPAA polypeptide or an isolated nucleic acid molecule encoding such animmunogenic PAA polypeptide. The polypeptide or nucleic acid vaccinesmay be used together with one or more adjuvants.

In yet other aspects, the present disclosure provides vaccine-basedimmunotherapy regimens (or “VBIR”) that involve co-administration of avaccine delivering various tumor associated antigens (TAAs) for inducingTAA specific immune responses to treat a variety of cancers incombination with at least one immune-suppressive-cell inhibitor and atleast one immune-effector-cell enhancer. Specifically, in some aspects,the disclosure provides a method of enhancing the immunogenicity ortherapeutic effect of a vaccine for the treatment of a neoplasticdisorder in a mammal, comprising administering to the mammal receivingthe vaccine an effective amount of at least one immune-suppressive-cellinhibitor and at least one immune-effector-cell enhancer. In a furtheraspect, the disclosure provides a method of treating a neoplasticdisorder in a mammal, comprising administering to the mammal a vaccine,at least one immune-suppressive-cell inhibitor, and at least oneimmune-effector-cell enhancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic illustration of PJV7563 vector.

FIG. 2. Amino acid alignment of five viral 2A cassettes. The skippedglycine-proline bonds are indicated by asterisks.

FIG. 3. Sequence of the preferred EMCV IRES. The translation initiationsite is indicated by the asterisk. The minimal IRES element excludes theunderlined first 5 codons of the EMCV L protein.

FIG. 4. Dot plots showing expression of the human PSMA modified antigen(amino acids 15-750) and full length human PSCA on the surface of HEK293cells transfected with dual antigen vaccine constructs as measured byflow cytometry.

FIGS. 5A and 5B. Image of Western blots showing expression of the humanPSMA modified antigen (amino acids 15-750; FIG. 5A) and full lengthhuman PSCA (FIG. 5B) in HEK293 cells transfected with dual antigenvaccine constructs as measured by western blotting with PSMA and PSCAspecific monoclonal antibodies.

FIG. 6. Image of Western blots showing expression of human PSA cytosolicantigen (amino acids 25-261) in HEK293 cells transfected with dualantigen vaccine constructs as measured by western blotting with a PSAspecific monoclonal antibody. Lane 5300 exhibited a faint band about 2kD larger than PSA, consistent with a C-terminal fusion of the 2Apeptide.

FIGS. 7A, 7B. Dot plots showing expression of human PSMA modifiedantigen (amino acids 15-750) and full length human PSCA on the surfaceof HEK293 cells transfected with either single promoter triple antigenconstructs (FIG. 7A) or dual promoter triple antigen vaccine constructs(FIG. 7B) as measured by flow cytometry.

FIGS. 8A, 8B. Images of Western blots showing expression of human PSA inHEK293 cells transfected with either single promoter triple antigenconstructs (FIG. 8A) or dual promoter triple antigen vaccine constructs(FIG. 8B) as measured by western blotting with a PSA specific monoclonalantibody. The bands in lanes 5259 and 456 are spillover from lane 5297.Although not visible in the scanned gel, lanes 456, 457, and 458exhibited a band about 2 kD larger than PSA.

FIGS. 9A-9D. Graphs depicting results of a representative study thatevaluates the immunogenicity of the triple antigen vaccines by IFN-γELISPOT assay, in which recognition of endogenous prostate antigens wasassessed by examining T cell responses to (a) TRAMP C2 cells expressingPSMA (FIG. 9A), (b) TRAMP C2 cells expressing PSCA (FIG. 9B), (c) TRAMPC2 cells expressing PSA (FIG. 9C), and (d) TRAMP C2 cells expressingPSMA, PSA, and PSCA (FIG. 9D).

FIGS. 10A-10D. Graphs depicting results of a representative study thatevaluates the immunogenicity of the triple antigen vaccines by IFN-γELISPOT assay, in which T cell responses to (a) individual PSMA peptides(FIG. 10A), (b) three PSMA peptide pools (FIG. 10B), (c) a PSCA peptide(FIG. 10C), and (d) PSA peptides (FIG. 10D) were assessed.

FIG. 11. Graph depicting results of a representative study thatevaluates the immunogenicity of the triple antigen vaccines by anti-PSMAantibody titers.

FIG. 12. Graph depicting results of a representative study thatevaluates the immunogenicity of the triple antigen vaccines by anti-PSCAantibody titers.

FIG. 13. Graph depicting results of a representative study thatevaluates the immunogenicity of the triple antigen vaccines by anti-PSMAantibody cell-surface binding.

FIG. 14. Graph depicting results of a representative study thatevaluates the immunogenicity of the triple antigen vaccines by anti-PSCAantibody cell-surface binding.

FIGS. 15A-15C. Graphs depicting results of a representative study thatevaluates the immunogenicity of the dual antigen vaccines by IFN-γELISPOT assay, in which recognition of endogenous prostate antigens wasassessed by examining T cell responses to (a) TRAMP C2 cells expressingPSMA (FIG. 15A), (b) TRAMP C2 cells expressing PSCA (FIG. 15B), and (c)TRAMP C2 cells expressing PSMA, PSA, and PSCA (FIG. 15C).

FIGS. 16A-16C. Graphs depicting results of a representative study thatevaluates the immunogenicity of the dual antigen vaccines by IFN-γELISPOT assay, in which T cell responses to (a) individual PSMA peptides(FIG. 16A), (b) three different PSMA peptide pools (FIG. 16B), and (c) aPSCA peptide (FIG. 16C) were assessed.

FIG. 17. Graph depicting results of a representative study thatevaluates the immunogenicity of the dual antigen vaccines by anti-PSMAantibody titers.

FIG. 18. Graph depicting results of a representative study thatevaluates the immunogenicity of the dual antigen vaccines by anti-PSCAantibody titers.

FIG. 19. Graph depicting results of a representative study thatevaluates the immunogenicity of the dual antigen vaccines by anti-PSMAantibody cell-surface binding.

FIG. 20. Graph depicting results of a representative study thatevaluates the immunogenicity of the dual antigen vaccines by anti-PSCAantibody cell-surface binding.

FIGS. 21A-21 D. Graphs depicting results of a representative study thatevaluates the immunogenicity of the dual antigen vaccines by IFN-γELISPOT assay, in which recognition of endogenous PSMA, PSCA, and PSAwas assessed by examining T cell responses to (a) TRAMP C2 cellsexpressing PSMA (FIG. 21A), (b) TRAMP C2 cells expressing PSCA (FIG.21B), (c) TRAMP C2 cells expressing PSA (FIG. 21C), and (d) TRAMP C2cells expressing PSMA, PSA, and PSCA (FIG. 21D).

FIG. 22. Graph depicting results of a representative study thatevaluates the immunogenicity of the dual antigen vaccines by anti-PSMAantibody titers.

FIG. 23. Graph depicting results of a representative study thatevaluates the immunogenicity of the dual antigen vaccines by anti-PSCAantibody titers.

FIG. 24. Graph depicting results of a representative study thatevaluates the immunogenicity of the dual antigen vaccines by anti-PSMAantibody cell-surface binding.

FIG. 25. Graph depicting results of a representative study thatevaluates the immunogenicity of the dual antigen vaccines by anti-PSCAantibody cell-surface binding.

FIG. 26. Graph depicting results of a representative study thatevaluates the T cell immune response elicited by human PSMA modifiedantigen (amino acids 15-750) versus full-length human PSMA (amino acids1-750) in C57BL/6 mice.

FIGS. 27A, 27B. Graphs depicting results of a representative study thatevaluates the T cell immune response of human PSMA modified antigen(amino acids 15-750) versus full-length human PSMA antigen (amino acids1-750) in Pasteur (HLA-A2/DR1) transgenic mice by IFN-γ ELISPOT assayusing (a) PSMA derived HLA-A2-restricted peptides (FIG. 27A) or (b)SK-Mel5 cells transduced with Ad-hPSMA or purified hPSMA full-lengthprotein (FIG. 27B).

FIG. 28. Graph depicting results of a representative study thatevaluates the immunogenicity of the human modified and full-length PSMAvaccines by anti-PSMA antibody titers.

FIG. 29. Graph depicting results of a representative study thatevaluates the immunogenicity of the human modified and full-length PSMAvaccines by anti-PSMA antibody cell-surface binding.

FIG. 30. Graph depicting results of a representative study thatevaluates the blood anti-CTLA-4 monoclonal antibody levels measured bycompetitive ELISA in Indian Rhesus macaques injected with anti-CTLA-4(CP-675, 206) at 10 mg/kg.

FIGS. 31A and 31B. Graphs depicting results of a representative studythat evaluates the immunomodulatory activity of anti-murine CTLA-4monoclonal antibody (clone 9H10) on the quality of the immune responsesinduced by a rat Her-2 DNA vaccine using an intracellular cytokinestaining assay, in which (a) cytokine positive CD8 T cells (FIG. 31A)and (b) cytokine positive CD4 T cells (FIG. 31B) were measured.

FIG. 32. Graph depicting results of a representative study thatevaluates the effect of sunitinib malate (Sutent) on the anti-tumorefficacy of a cancer vaccine (rHER2) in mice, in which the subcutaneoustumor growth rate was measured.

FIGS. 33A-33D. Graphs depicting results from a representative study thatevaluates the effect of sunitinib malate (Sutent) on the anti-tumorefficacy of a cancer vaccine (rHER2), in which individual tumor growthrates were measured in mice treated with (a) the control agents (FIG.33A), (b) sunitinib malate and the control vaccine (FIG. 33B), (c) thevehicle and the cancer vaccine (FIG. 33C), or (d) sunitinib malate andthe cancer vaccine (FIG. 33D).

FIG. 34. Graph showing the Kaplan-Meier survival curves of the groups ofmice from the study described in FIGS. 33A-33D that evaluates the effectof sunitinib malate (Sutent) on the anti-tumor efficacy of a cancervaccine (rHER2).

FIGS. 35A, 35B. Graphs showing changes in myeloid derived suppressorcells (Gr1+CD11b+; FIG. 35A) and Treg containing CD25+CD4+ cells (FIG.35B) in the periphery blood of the groups of mice from the studydescribed FIGS. 33A-33D.

FIGS. 36A-36C. Graphs depicting results of a representative study in amouse tumor model that evaluates the effect of sunitinib malate (Sutent)on the total number of (a) Tregs (CD4+CD25+Foxp3+; FIG. 36A), (b)myeloid derived suppressor cells (Gr1+CD11b+; FIG. 36B), and (c)PD-1+CD8 T cells (FIG. 36C) isolated from tumors of the mice.

FIG. 37. Graph showing the Kaplan-Meier survival curves of the groups ofmice from a representative study evaluating the effect of sunitinibmalate (Sutent) and an anti-murine CTLA-4 monoclonal antibody (clone9D9) on the anti-tumor efficacy of a cancer vaccine (vaccine) insubcutaneous TUBO tumor bearing BALB/neuT mice.

FIG. 38. Graph showing kinetics of the blood sunitinib levels ofBALB/neuT mice with subcutaneous TUBO tumors.

FIG. 39. Graph showing the Kaplan-Meier survival curves of the groups ofmice from a representative study that evaluates the effect of sunitinibmalate (Sutent) on the anti-tumor efficacy of a cancer vaccine inBALB/neuT mice with subcutaneous TUBO tumors.

FIG. 40. Graph depicting the IFNγ ELISPOT results from a representativestudy evaluating the effect of CpG7909 and an anti-CD40 antibody(Bioxcell #BE0016-2) on the antigen specific T cell responses induced bya cancer vaccine (rHER2).

FIGS. 41A, 41B. Graphs depicting results of a representative study thatevaluates the immunomodulatory activity of CpG7909 on the quality of theimmune responses induced by a cancer vaccine (PMED) using intracellularcytokine staining assay, in which cytokine positive CD8 T cells (FIG.41A) and cytokine positive CD4 T cells (FIG. 41B) were measured.

FIGS. 42A, 42B. Graphs depicting results of a representative study thatevaluates the immunomodulatory activity of an agonistic anti-murine CD40monoclonal antibody on the quality of the immune responses induced by acancer vaccine (PMED) using intracellular cytokine staining assay, inwhich cytokine positive CD8 T cells (FIG. 42A) and cytokine positive CD4T cells (FIG. 42B) were measured.

FIG. 43. Graph showing the Kaplan-Meier survival curves of the groups ofmice from a representative study that evaluates the effect of low dosesunitinib malate (Sutent) on the anti-tumor efficacy of a cancer vaccinein spontaneous mammary tumor bearing BALB/neuT mice.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

The term “adjuvant” refers to a substance that is capable of enhancing,accelerating, or prolonging an immune response when given with a vaccineimmunogen.

The term “agonist” refers to is a substance which promotes (induces,causes, enhances or increases) the activity of another molecule or areceptor. The term agonist encompasses substances which bind receptor(e.g., an antibody, a homolog of a natural ligand from another species)and substances which promote receptor function without binding thereto(e.g., by activating an associated protein).

The term “antagonist” or “inhibitor” refers to a substance thatpartially or fully blocks, inhibits, or neutralizes a biologicalactivity of another molecule or receptor.

The term “co-administration” refers to administration of two or moreagents to the same subject during a treatment period. The two or moreagents may be encompassed in a single formulation and thus beadministered simultaneously. Alternatively, the two or more agents maybe in separate physical formulations and administered separately, eithersequentially or simultaneously to the subject. The term “administeredsimultaneously” or “simultaneous administration” means that theadministration of the first agent and that of a second agent overlap intime with each other, while the term “administered sequentially” or“sequential administration” means that the administration of the firstagent and that of a second agent does not overlap in time with eachother.

The term “conserved T cell epitope” refers to one of the following aminoacid sequences of the human PSMA protein as set forth in SEQ ID NO. 1:

amino acids 168-176 (GMPEGDLVY),

amino acids 347-356 (HSTNGVTRIY),

amino acids 557-566 (ETYELVEKFY),

amino acids 207-215 (KVFRGNKVK),

amino acids 431-440 (STEWAEENSR),

amino acids 4-12 (LLHETDSAV),

amino acids 27-35 (VLAGGFFLL),

amino acids 168-177 (GMPEGDLVYV),

amino acids 441-450 (LLQERGVAYI),

amino acids 469-477 (LMYSLVHNL),

amino acids 711-719 (ALFDIESKV),

amino acids 663-671 (MNDQVMFL),

amino acids 178-186 (NYARTEDFF),

amino acids, 227-235 (LYSDPADYF),

amino acids 624-632 (TYSVSFDSL),

amino acids 334-348 (TGNFSTQKVKMHIHS),

amino acids 459-473 (NYTLRVDCTPLMYSL),

amino acids 687-701(YRHVIYAPSSHNKYA), and

amino acids 730-744 (RQIYVAAFTVQAAAE).

The term “cytosolic” means that after a nucleotide sequence encoding aparticular polypeptide is expressed by a host cell, the expressedpolypeptide is retained inside the host cell.

The terms “degenerate variant” refers to DNA sequences that havesubstitutions of bases but encode the same polypeptide.

The term “effective amount” refers to an amount administered to a mammalthat is sufficient to cause a desired effect in the mammal.

The term “fragment” of a given polypeptide refers to a polypeptide thatis shorter than the given polypeptide and shares 100% identity with thesequence of the given polypeptide.

The term “identical” or percent “identity,” in the context of two ormore nucleic acid or polypeptide sequences, refers to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence.

The term “immune-effector-cell enhancer” or “IEC enhancer” refers to asubstance capable of increasing or enhancing the number, quality, orfunction of one or more types of immune effector cells of a mammal.Examples of immune effector cells include cytolytic CD8 T cells, CD40 Tcells, NK cells, and B cells.

The term “immune modulator” refers to a substance capable of altering(e.g., inhibiting, decreasing, increasing, enhancing or stimulating) theworking of any component of the innate, humoral or cellular immunesystem of a mammal. Thus, the term “immune modulator” encompasses the“immune-effector-cell enhancer” as defined herein and the“immune-suppressive-cell inhibitor” as defined herein, as well assubstance that affects other components of the immune system of amammal.

The term “immune response” refers to any detectable response to aparticular substance (such as an antigen or immunogen) by the immunesystem of a host vertebrate animal, including, but not limited to,innate immune responses (e.g., activation of Toll receptor signalingcascade), cell-mediated immune responses (e.g., responses mediated by Tcells, such as antigen-specific T cells, and non-specific cells of theimmune system), and humoral immune responses (e.g., responses mediatedby B cells, such as generation and secretion of antibodies into theplasma, lymph, and/or tissue fluids). Examples of immune responsesinclude an alteration (e.g., increase) in Toll-like receptor activation,lymphokine (e.g., cytokine (e.g., Th1, Th2 or Th17 type cytokines) orchemokine) expression or secretion, macrophage activation, dendriticcell activation, T cell (e.g., CD4+ or CD8+T cell) activation, NK cellactivation, B cell activation (e.g., antibody generation and/orsecretion), binding of an immunogen (e.g., antigen (e.g., immunogenicpolypolypeptide)) to an MHC molecule, induction of a cytotoxic Tlymphocyte (“CTL”) response, induction of a B cell response (e.g.,antibody production), and, expansion (e.g., growth of a population ofcells) of cells of the immune system (e.g., T cells and B cells), andincreased processing and presentation of antigen by antigen presentingcells. The term “immune response” also encompasses any detectableresponse to a particular substance (such as an antigen or immunogen) byone or more components of the immune system of a vertebrate animal invitro.

The term “immunogenic” refers to the ability of a substance to cause,elicit, stimulate, or induce an immune response, or to improve, enhance,increase or prolong a pre-existing immune response, against a particularantigen, whether alone or when linked to a carrier, in the presence orabsence of an adjuvant.

The term “immunogenic PSA polypeptide” refers to a polypeptide that isimmunogenic against human PSA protein or against cells expressing humanPSA protein.

The term “immunogenic PSCA polypeptide” refers to a polypeptide that isimmunogenic against human PSCA protein or against cells expressing humanPSCA protein.

The term “immunogenic PSMA polypeptide” refers to a polypeptide that isimmunogenic against human PSMA protein or against cells expressing humanPSMA protein.

The term “immunogenic PAA polypeptide” refers to an “immunogenic PSApolypeptide,” an “immunogenic PSCA polypeptide,” or an “immunogenic PSMApolypeptide” as defined herein above.

The term “immunogenic PSA nucleic acid molecule” refers to a nucleicacid molecule that encodes an immunogenic PSA polypeptide as definedherein.

The term “immunogenic PSCA nucleic acid molecule” refers to a nucleicacid molecule that encodes an “immunogenic PSCA polypeptide” as definedherein.

The term “immunogenic PSMA nucleic acid molecule” refers to a nucleicacid molecule that encodes an “immunogenic PSMA polypeptide” as definedherein.

The term “immunogenic PAA nucleic acid molecule” refers to a nucleicacid molecule that encodes an “immunogenic PSA polypeptide,” an“immunogenic PSCA polypeptide,” or an “immunogenic PSMA polypeptide” asdefined herein above.

The term “immune-suppressive-cell inhibitor” or “ISC inhibitor” refersto a substance capable of reducing or suppressing the number or functionof immune suppressive cells of a mammal. Examples of immune suppressivecells include regulatory T cells (“T regs”), myeloid-derived suppressorcells, and tumor-associated macrophages.

The term “intradermal administration,” or “administered intradermally,”in the context of administering a substance, such as a therapeutic agentor an immune modulator, to a mammal including a human, refers to thedelivery of the substance into the dermis layer of the skin of themammal. The skin of a mammal is composed of three layers—the epidermis,dermis, and subcutaneous layer. The epidermis is the relatively thin,tough, outer layer of the skin. Most of the cells in the epidermis arekeratinocytes. The dermis, the skin's next layer, is a thick layer offibrous and elastic tissue (made mostly of collagen, elastin, andfibrillin) that gives the skin its flexibility and strength. The dermiscontains nerve endings, sweat glands and oil (sebaceous) glands, hairfollicles, and blood vessels. The dermis varies in thickness dependingon the location of the skin. In humans it is about 0.3 mm on the eyelidand about 3.0 mm on the back. The subcutaneous layer is made up of fatand connective tissue that houses larger blood vessels and nerves. Thethickness of this layer varies throughout the body and from person toperson. The term “intradermal administration” refers to delivery of asubstance to the inside of the dermis layer. In contrast; “subcutaneousadministration” refers to the administration of a substance into thesubcutaneous layer and “topical administration” refers to theadministration of a substance onto the surface of the skin.

The term “local administration” or “administered locally” encompasses“topical administration,” “intradermal administration,” and“subcutaneous administration,” each as defined herein above. This termalso encompasses “intratumoral administration,” which refers toadministration of a substance to the inside of a tumor. Localadministration is intended to allow for high local concentrations aroundthe site of administration for a period of time until systemicbiodistribution has been achieved with of the administered substance,while “systemic administration” is intended for the administeredsubstance to be absorbed into the blood and attain systemic exposurerapidly by being distributed through the circulatory system to organs ortissues throughout the body.

The term “mammal” refers to any animal species of the Mammalia class.Examples of mammals include: humans; non-human primates such as monkeys;laboratory animals such as rats, mice, guinea pigs; domestic animalssuch as cats, dogs, rabbits, cattle, sheep, goats, horses, and pigs; andcaptive wild animals such as lions, tigers, elephants, and the like.

The term “membrane-bound” means that after a nucleotide sequenceencoding a particular polypeptide is expressed by a host cell, theexpressed polypeptide is bound to, attached to, or otherwise associatedwith, the membrane of the cell.

The term “neoplastic disorder” refers to a condition in which cellsproliferate at an abnormally high and uncontrolled rate, the rateexceeding and uncoordinated with that of the surrounding normal tissues.It usually results in a solid lesion or lump known as “tumor.” This termencompasses benign and malignant neoplastic disorders. The term“malignant neoplastic disorder”, which is used interchangeably with theterm “cancer” in the present disclosure, refers to a neoplastic disordercharacterized by the ability of the tumor cells to spread to otherlocations in the body (known as “metastasis”). The term “benignneoplastic disorder” refers to a neoplastic disorder in which the tumorcells lack the ability to metastasize.

The term “operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a transgene is ligated in such a way that expression of the transgeneis achieved under conditions compatible with the control sequences.

The term “ortholog” refers to genes in different species that aresimilar to each other and originated from a common ancestor.

The term “pharmaceutically acceptable excipient” refers to a substancein an immunogenic or vaccine composition, other than the activeingredients (e.g., the antigen, antigen-coding nucleic acid, immunemodulator, or adjuvant) that is compatible with the active ingredientsand does not cause significant untoward effect in subjects to whom it isadministered.

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably herein, and refer to a polymeric form of amino acids ofany length, which can include coded and non-coded amino acids,chemically, or biochemically modified or derivatized amino acids, andpolypeptides having modified polypeptide backbones.

The term “preventing” or “prevent” refers to (a) keeping a disorder fromoccurring or (b) delaying the onset of a disorder or onset of symptomsof a disorder.

The term “prostate-associated-antigen” (or PAA) refers to the TAA (asdefined herein) that is specifically expressed on prostate tumor cellsor expressed at a higher frequency or density by tumor cells than bynon-tumor cells of the same tissue type. Examples of PAA include PSA,PSCA, and PSMA.

The term “secreted” in the context of a polypeptide means that after anucleotide sequence encoding the polypeptide is expressed by a hostcell, the expressed polypeptide is secreted outside of the host cell.

The term “suboptimal dose” when used to describe the amount of an immunemodulator, such as a protein kinase inhibitor, refers to a dose of theimmune modulator that is below the minimum amount required to producethe desired therapeutic effect for the disease being treated when theimmune modulator is administered alone to a patient.

The term “treating,” “treatment,” or “treat” refers to abrogating adisorder, reducing the severity of a disorder, or reducing the severityor occurrence frequency of a symptom of a disorder.

The term “tumor-associated antigen” or “TAA” refers to an antigen whichis specifically expressed by tumor cells or expressed at a higherfrequency or density by tumor cells than by non-tumor cells of the sametissue type. Tumor-associated antigens may be antigens not normallyexpressed by the host; they may be mutated, truncated, misfolded, orotherwise abnormal manifestations of molecules normally expressed by thehost; they may be identical to molecules normally expressed butexpressed at abnormally high levels; or they may be expressed in acontext or milieu that is abnormal. Tumor-associated antigens may be,for example, proteins or protein fragments, complex carbohydrates,gangliosides, haptens, nucleic acids, or any combination of these orother biological molecules.

The term “vaccine” refers to an immunogenic composition foradministration to a mammal for eliciting an immune response against aparticular antigen.

The term “variant” of a given polypeptide refers to a polypeptide thatshares less than 100% but more than 80% identity to the amino acidsequence of that given polypeptide and exhibits at least some of theimmunogenic activity of that given polypeptide.

The term “vector” refers to a nucleic acid molecule capable oftransporting or transferring a foreign nucleic acid molecule. The termencompasses both expression vectors and transcription vectors. The term“expression vector” refers to a vector capable of expressing the insertin the target cell, and generally contain control sequences, such asenhancer, promoter, and terminator sequences, that drive expression ofthe insert. The term “transcription vector” refers to a vector capableof being transcribed but not translated. Transcription vectors are usedto amplify their insert. The foreign nucleic acid molecule is referredto as “insert” or “transgene.” A vector generally consists of an insertand a larger sequence that serves as the backbone of the vector. Basedon the structure or origin of vectors, major types of vectors includeplasmid vectors, cosmid vectors, phage vectors such as lambda phage,viral vectors such as adenovirus (Ad) vectors, and artificialchromosomes.

B. Immunogenic Prostate-Associated-Antigen (PAA) Polypeptides

In some aspects, the present disclosure provides isolated immunogenicPSA polypeptides and PSMA polypeptides, which are useful, for example,for eliciting an immune response in vivo (e.g. in an animal, includinghumans) or in vitro, activating effector T cells, or generatingantibodies specific for PSA and PSMA, respectively, or for use as acomponent in vaccines for treating cancer, particularly prostate cancer.These polypeptides can be prepared by methods known in the art in lightof the present disclosure. The capability of the polypeptides to elicitan immune response can be measured in in vitro assays or in vivo assays.In vitro assays for determining the capability of a polypeptide or DNAconstruct to elicit immune responses are known in the art. One exampleof such in vitro assays is to measure the capability of the polypeptideor nucleic acid expressing an polypeptide to stimulate T cell responseas described in U.S. Pat. No. 7,387,882, the disclosure of which isincorporated in this application. The assay method comprises the stepsof: (1) contacting antigen presenting cells in culture with an antigenthereby the antigen can be taken up and processed by the antigenpresenting cells, producing one or more processed antigens; (2)contacting the antigen presenting cells with T cells under conditionssufficient for the T cells to respond to one or more of the processedantigens; (3) determining whether the T cells respond to one or more ofthe processed antigens. The T cells used may be CD8⁺ T cells or CD4⁺ Tcells. T cell response may be determined by measuring the release of oneof more of cytokines, such as interferon-gamma and interleukin-2, lysisof the antigen presenting cells (tumor cells), and production ofantibodies by B cells.

B-1. Immunogenic PSMA Polypeptides

In one aspect, the present disclosure provides isolated immunogenic PSMApolypeptides which have at least 90% identity to amino acids 15-750 ofthe human PSMA of SEQ ID NO:1 and comprise the amino acids of at least10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the conserved T cellepitopes of the human PSMA at corresponding positions.

In some embodiments, the immunogenic PSMA polypeptides comprise at least15, 16, 17, 18, or 19 of the conserved T cell epitopes of the humanPSMA.

In some embodiments, the present disclosure provides an immunogenic PSMApolypeptide consisting of the amino acid sequence of SEQ ID NO:9, or animmunogenic PSMA polypeptide having 93%-99%, 94%-98%, or 94%-97%identity to the amino acid sequence of SEQ ID NO:9.

Examples of some particular immunogenic PSMA polypeptides include:

1) a polypeptide consisting of amino acids 15-750 of SEQ ID NO: 1;

2) a polypeptide comprising the amino acids 4-739 of SEQ ID NO: 3;

3) a polypeptide comprising the amino acids 4-739 of SEQ ID NO:5;

4) a polypeptide comprising the amino acids 4-739 of SEQ ID NO:7;

2) a polypeptide comprising the amino acid sequence of SEQ ID NO:3;

3) a polypeptide comprising the amino acid sequence of SEQ ID NO:5; and

4) a polypeptide comprising the amino acid sequence of SEQ ID NO:7.

In other embodiments, the present disclosure provides an immunogenicPSMA polypeptide selected from the group consisting of:

1) a polypeptide consisting of the amino acid sequence of SEQ ID NO:11

2) a polypeptide consisting of the amino acid sequence of SEQ ID NO:13;and

3) a polypeptide comprising the amino acid sequence of SEQ ID NO:13.

In some other embodiments, the present disclosure provides isolatedimmunogenic PSMA polypeptides that are variants of any of the followingpolypeptides:

2) a polypeptide comprising the amino acids 4-739 of SEQ ID NO: 3;

3) a polypeptide comprising the amino acids 4-739 of SEQ ID NO: 5; and

4) a polypeptide comprising the amino acids 4-739 of SEQ ID NO: 7,wherein the amino acid sequence of the variant has 93%-99% identity tothe sequence of SEQ ID NO:1 and share at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% identity with the amino acidsequence of SEQ ID NO: 3, 5, or 7.

The variants of a given PAA polypeptide can be obtained by deleting,inserting, or substituting one or more amino acids in the parentimmunogenic PAA polypeptide. An example for the production of suchvariants is the conservative substitution of individual amino acids ofthe polypeptides, that is, by substituting one amino acid for anotherhaving similar properties.

An immunogenic PSMA polypeptide of the invention may be constructed byconserving some or all of the conserved T cell epitopes of the humanPSMA of SEQ ID NO:1 while substituting certain amino acids in theremaining regions of the human PSMA with amino acids found in one ormore orthologs of human PSMA at corresponding positions. Sequences ofvarious PSMA orthologs that may be utilized to make the immunogenic PSMApolypeptides are available from the GeneBank database. These orthologsalong with their NCBI ID numbers are provided in Table 18. Substitutionsof amino acids of human PSMA with amino acids from one or more of theorthologs may be conservative substitutions or non-conservativesubstitutions, or both, and may be selected based on a number of factorsknown in the art, including the divergence needed to be achieved, MHCbinding, the presence of ortholog amino acids at the site ofsubstitution, surface exposure, and maintaining the 3-D structure of theprotein for optimal processing and presentation.

B-2. Immunogenic PSA Polypeptides

In another aspect, the present disclosure provides isolated immunogenicPSA polypeptides. In one embodiment, the isolated immunogenic PSApolypeptide is a polypeptide consisting of the amino acid sequence ofSEQ ID NO:15 or amino acids 4-263 of SEQ ID NO: 15, or a variantthereof. In another embodiment, the isolated immunogenic PSA polypeptideis a polypeptide consisting of the amino acid sequence of SEQ ID NO:17or amino acids 4-240 of SEQ ID NO: 17, or a variant thereof. In afurther embodiment, the isolated immunogenic PSA polypeptide is apolypeptide consisting of the amino acid sequence of SEQ ID NO:19 oramino acids 4-281 of SEQ ID NO: 19, or a variant thereof.

C. Nucleic Acid Molecules Encoding Immunogenic PAA Polypeptides

In some aspects, the present disclosure provides nucleic acid moleculesthat encode immunogenic PAA polypeptides. The nucleic acid molecules canbe deoxyribonucleotides (DNA) or ribonucleotides (RNA). Thus, a nucleicacid molecule can comprise a nucleotide sequence disclosed hereinwherein thymidine (T) can also be uracil (U), which reflects thedifferences between the chemical structures of DNA and RNA. The nucleicacid molecules can be modified forms, single or double stranded forms,or linear or circular forms. The nucleic acid molecules can be preparedusing methods known in the art light of the present disclosure.

C-1. Nucleic Acid Molecules Encoding Immunogenic PSMA Polypeptides

In one aspect, the present disclosure provides isolated nucleic acidmolecules, or degenerate variants thereof, which comprise a nucleotidesequence encoding an immunogenic PSMA polypeptide, including theimmunogenic PSMA polypeptides provided by the present disclosure or afunctional variant thereof

In some embodiments, the nucleotide sequence encodes a membrane-boundimmunogenic PSMA polypeptide. In some particular embodiments, theisolated nucleic acid molecule comprises a nucleotide sequence, or adegenerate variant thereof, selected from the group consisting of:

1) a nucleotide sequence encoding the amino acid sequence of SEQ IDNO:9;

2) a nucleotide sequence encoding amino acids 4-739 of SEQ ID NO:3;

3) a nucleotide sequence encoding amino acids 4-739 of SEQ ID NO:5; and

4) a nucleotide sequence encoding amino acids 4-739 of SEQ ID NO:7.

In some other particular embodiments, the nucleotide sequence encodes avariant of an immunogenic PSMA polypeptide of SEQ ID NO:3, SEQ ID NO:5,SEQ ID NO:7, or SEQ ID NO:9, wherein the variant has an amino acidsequence that has (a) 93% to 99% identity with the amino acid sequenceof SEQ ID NO:1 and (b) at least 80%, at least 85%, at least 90%, atleast 95%, or at least 99% identity with the amino acid sequence of SEQID NO: 3, 5, or 7.

In still some other particular embodiments, the isolated nucleic acidmolecule comprises a nucleotide sequence, or a degenerate variantthereof, selected from the group consisting of:

1) a nucleotide sequence comprising nucleotides 10-2217 of SEQ ID NO:4;

2) a nucleotide sequence comprising nucleotides 10-2217 of SEQ ID NO:6;

3) a nucleotide sequence comprising nucleotides 10-2217 of SEQ ID NO:8;and

4) a nucleotide sequence comprising nucleotides 10-2217 of SEQ ID NO:10.

C-2. Nucleic Acid Molecules Encoding Immunogenic PSA Polypeptides

In another aspect, the present disclosure provides isolated nucleic acidmolecules, or degenerate variants thereof, which encode an immunogenicPSA polypeptide, including the immunogenic PSA polypeptides provided bythe present disclosure.

In some embodiments, the isolated nucleic acid molecule comprises orconsists of a nucleotide sequence encoding a cytosolic immunogenic PSApolypeptide. In one embodiment, the nucleotide sequence encodes acytosolic immunogenic PSA polypeptide consisting of consecutive aminoacid residues 4-240 of SEQ ID NO:17. In another embodiment, thenucleotide sequence encodes a cytosolic immunogenic PSA polypeptidecomprising the amino acid sequence of SEQ ID NO:17. In still anotherembodiment, the nucleotide sequence encodes a cytosolic immunogenic PSApolypeptide consisting of the amino acid sequence of SEQ ID NO:17. Inyet another embodiment, the nucleotide sequence encodes a functionalvariant of any of said cytosolic immunogenic polypeptides providedherein above.

In some other embodiments, the isolated nucleic acid molecule comprisesa nucleotide sequence encoding a membrane-bound immunogenic PSApolypeptide. In one embodiment, the nucleotide sequence encodes amembrane-bound immunogenic PSA polypeptide comprising consecutive aminoacid residues 4-281 of SEQ ID NO:19. In another embodiment, thenucleotide sequence encodes a membrane-bound immunogenic PSA polypeptidecomprising the amino acid sequence of SEQ ID NO:19. In still anotherembodiment, the nucleotide sequence encodes a membrane-bound immunogenicPSA polypeptide consisting of the amino acid sequence of SEQ ID NO:19.In yet other embodiments, the nucleotide sequence encodes a functionalvariant of any of said membrane-bound immunogenic PSA polypeptidesprovided herein above.

Examples of particular nucleic acid molecules provided by the presentdisclosure include:

1) a nucleic acid molecule consisting of the nucleotide sequence of SEQID NO: 18;

-   -   2) a nucleic acid molecule comprising the nucleotide sequence of        SEQ ID NO; 18;

3) a nucleic acid molecule consisting of the nucleotide sequence of SEQID NO; 20;

4) a nucleic acid molecule comprising the nucleotide sequence of SEQ IDNO: 20; and

5) a degenerate variant of any of the nucleic acid molecules 1)-4)above.

C-3. Nucleic Acid Molecules Encoding Two or More Immunogenic PAAPolypeptides

In another aspect, the present disclosure provides a nucleic acidmolecule that encodes more than one immunogenic PAA polypeptide, forexample at least two, at least three, or at least four immunogenic PAApolypeptides. Such nucleic acid molecules are also be referred to as“multi-antigen constructs,” “multi-antigen vaccine,” “multi-antigenplasmid,” and the like, in the present disclosure. Thus, in someaspects, one nucleic acid molecule carries two coding nucleotidesequences wherein each of the coding nucleotide sequences expresses anindividual immunogenic PAA polypeptide. Such a nucleic acid molecule isalso referred to as “dual antigen construct,” “dual antigen vaccine,” or“dual antigen plasmid,” etc., in this disclosure. In some other aspects,one nucleic acid molecule carries three coding nucleotide sequenceswherein each of the coding nucleotide sequences expresses an individualimmunogenic PAA polypeptide. Such a nucleic acid molecule is alsoreferred to as “triple antigen construct,” “triple antigen vaccine,” or“triple antigen plasmid” in this disclosure. The individual PAApolypeptides encoded by a multi-antigen construct may be immunogenicagainst the same antigen, such as PSMA, PSA, or PSCA. The individual PAApolypeptides encoded by a multi-antigen construct may be immunogenicagainst different antigens, for example, one PAA polypeptide being aPSMA polypeptide and another one a PSA polypeptide. Specifically, onemulti-antigen construct may encode two or more immunogenic PAApolypeptides in any one of the following combinations:

1) at least one immunogenic PSMA polypeptide and at least oneimmunogenic PSA polypeptide;

2) at least one immunogenic PSMA polypeptide and at least oneimmunogenic PSCA polypeptide;

3) at least one immunogenic PSA polypeptide and at least one immunogenicPSCA polypeptide; and

4) at least one immunogenic PSMA polypeptide, at least one immunogenicPSA polypeptide, and at least one immunogenic PSCA polypeptide.

The immunogenic PSMA polypeptides encoded by a multi-antigen constructmay be either cytosolic, secreted, or membrane-bound, but preferablymembrane-bound. Similarly, the immunogenic PSA polypeptide encoded by amulti-antigen construct may be either cytosolic, secreted, ormembrane-bound, but preferably cytosolic. The immunogenic PSCApolypeptide encoded by a multi-antigen construct is preferably the fulllength human PSCA protein, the amino acid sequence of which is set forthin SEQ ID No:21.

In some embodiments, the present disclosure provides a multi-antigenconstruct that encodes at least one membrane-bound immunogenic PSMApolypeptide and at least one membrane-bound immunogenic PSA polypeptide.

In some other embodiments, the present disclosure provides amulti-antigen construct that encodes at least one membrane-boundimmunogenic PSMA polypeptide, at least one cytosolic immunogenic PSApolypeptide, and at least one immunogenic PSCA polypeptide, wherein theat least one cytosolic immunogenic PSA polypeptide comprises amino acids4-240 of SEQ ID NO:17, wherein the at least one immunogenic PSCApolypeptide is the full length human PSCA protein of SEQ ID NO:21, andwherein the at least one immunogenic PSMA polypeptide is selected fromthe group consisting of:

1) a polypeptide comprising amino acids 15-750 of SEQ ID NO: 1;

2) a polypeptide comprising the amino acid sequence of SEQ ID NO:3;

3) a polypeptide comprising the amino acid sequence of SEQ ID NO:5;

4) a polypeptide comprising the amino acid sequence of SEQ ID NO:7;

5) a polypeptide comprising the amino acids 4-739 of SEQ ID NO:9;

6) a polypeptide comprising the amino acids 4-739 of SEQ ID NO:3;

7) a polypeptide comprising the amino acids 4-739 of SEQ ID NO:5;

8) a polypeptide comprising the amino acids 4-739 of SEQ ID NO:7; and

9) polypeptide comprising the amino acid sequence of SEQ ID NO: 9.

In some particular embodiments, the present disclosure provides amulti-antigen construct comprising at least one nucleotide sequenceencoding an immunogenic PSMA polypeptide, at least one nucleotidesequence encoding an immunogenic PSA polypeptide, and at least onenucleotide sequence encoding an immunogenic PSCA polypeptide, whereinthe nucleotide sequence encoding the immunogenic PSA polypeptide isselected from the nucleotide sequence of SEQ ID NO: 18 or SEQ ID NO: 20,wherein the nucleotide sequence encoding the immunogenic PSCApolypeptide is set forth in SEQ ID NO:22, and wherein the nucleotidesequence encoding the immunogenic PSMA polypeptide is selected from thegroup consisting of:

1) the nucleotide sequence of SEQ ID NO:2;

2) the nucleotide sequence of SEQ ID NO:4;

3) the nucleotide sequence of SEQ ID NO:6;

4) the nucleotide sequence of SEQ ID NO:8;

5) the nucleotide sequence of SEQ ID NO:10;

6) a nucleotide sequence comprising nucleotides 10-2217 of SEQ ID NO:4;

7) a nucleotide sequence comprising nucleotides 10-2217 of SEQ ID NO:6;

8) a nucleotide sequence comprising nucleotides 10-2217 of SEQ ID NO:8;and

9) a nucleotide sequence comprising nucleotides 10-2217 of SEQ ID NO:10.

Examples of specific multi-antigen constructs provided by the presentdisclosure include the nucleic acid molecules that comprise a nucleotidesequence set forth in SEQ ID NOs:23-36.

Multi-antigen constructs provided by the present disclosure can beprepared using various techniques known in the art in light of thedisclosure. For example, a multi-antigen construct can be constructed byincorporating multiple independent promoters into a single plasmid(Huang, Y., Z. Chen, et al. (2008). “Design, construction, andcharacterization of a dual-promoter multigenic DNA vaccine directedagainst an HIV-1 subtype C/B′ recombinant.” J Acquir Immune Defic Syndr47(4): 403-411; Xu, K., Z. Y. Ling, et al. (2011). “Broad humoral andcellular immunity elicited by a bivalent DNA vaccine encoding HA and NPgenes from an H5N1 virus.” Viral Immunol 24(1): 45-56). The plasmid canbe engineered to carry multiple expression cassettes, each consisting ofa) a eukaryotic promoter for initiating RNA polymerase dependenttranscription, with or without an enhancer element, b) a gene encoding atarget antigen, and c) a transcription terminator sequence. Upondelivery of the plasmid to the transfected cell nucleus, transcriptionwill be initiated from each promoter, resulting in the production ofseparate mRNAs, each encoding one of the target antigens. The mRNAs willbe independently translated, thereby producing the desired antigens.

Multi-antigen constructs provided by the present disclosure can also beconstructed using a single vector through the use of viral 2A-likepolypeptides (Szymczak, A. L. and D. A. Vignali (2005). “Development of2A peptide-based strategies in the design of multicistronic vectors.”Expert Opin Biol Ther 5(5): 627-638; de Felipe, P., G. A. Luke, et al.(2006). “E unum pluribus: multiple proteins from a self-processingpolyprotein.” Trends Biotechnol 24(2): 68-75; Luke, G. A., P. de Felipe,et al. (2008). “Occurrence, function and evolutionary origins of‘2A-like’ sequences in virus genomes.” J Gen Virol 89 (Pt 4): 1036-1042;Ibrahimi, A., G. Vande Velde, et al. (2009). “Highly efficientmulticistronic lentiviral vectors with peptide 2A sequences.” Hum GeneTher 20(8): 845-860; Kim, J. H., S. R. Lee, et al. (2011). “Highcleavage efficiency of a 2A peptide derived from porcine teschovirus-1in human cell lines, zebrafish and mice.” PLoS One 6(4): e18556). Thesepolypeptides, also called cleavage cassettes or CHYSELs (cis-actinghydrolase elements), are approximately 20 amino acids long with a highlyconserved carboxy terminal D-V/I-EXNPGP motif (FIG. 2). The cassettesare rare in nature, most commonly found in viruses such asFoot-and-mouth disease virus (FMDV), Equine rhinitis A virus (ERAV),Encephalomyocarditis virus (EMCV), Porcine teschovirus (PTV), and Thoseaasigna virus (TAV) (Luke, G. A., P. de Felipe, et al. (2008).“Occurrence, function and evolutionary origins of ‘2A-like’ sequences invirus genomes.” J Gen Virol 89 (Pt 4): 1036-1042). With a 2A-basedmulti-antigen expression strategy, genes encoding multiple targetantigens can be linked together in a single open reading frame,separated by 2A cassettes. The entire open reading frame can be clonedinto a vector with a single promoter and terminator. Upon delivery ofthe constructs to a host cell, mRNA encoding the multiple antigens willbe transcribed and translated as a single polyprotein. Duringtranslation of the 2A cassettes, ribosomes skip the bond between theC-terminal glycine and proline. The ribosomal skipping acts like acotranslational autocatalytic “cleavage” that releases upstream fromdownstream proteins. The incorporation of a 2A cassette between twoprotein antigens results in the addition of ˜20 amino acids onto theC-terminus of the upstream polypeptide and 1 amino acid (proline) to theN-terminus of downstream protein. In an adaptation of this methodology,protease cleavage sites can be incorporated at the N terminus of the 2Acassette such that ubiquitous proteases will cleave the cassette fromthe upstream protein (Fang, J., S. Yi, et al. (2007). “An antibodydelivery system for regulated expression of therapeutic levels ofmonoclonal antibodies in vivo.” Mol Ther 15(6): 1153-1159).

Another strategy for constructing the multi-antigen constructs providedby the present disclosure involves the use of an internal ribosomalentry site, or IRES. Internal ribosomal entry sites are RNA elements(FIG. 3) found in the 5′ untranslated regions of certain RNA molecules(Bonnal, S., C. Boutonnet, et al. (2003). “IRESdb: the Internal RibosomeEntry Site database.” Nucleic Acids Res 31(1): 427-428). They attracteukaryotic ribosomes to the RNA to facilitate translation of downstreamopen reading frames. Unlike normal cellular 7-methylguanosinecap-dependent translation, IRES-mediated translation can initiate at AUGcodons far within an RNA molecule. The highly efficient process can beexploited for use in multi-cistronic expression vectors (Bochkov, Y. A.and A. C. Palmenberg (2006). “Translational efficiency of EMCV IRES inbicistronic vectors is dependent upon IRES sequence and gene location.”Biotechniques 41(3): 283-284, 286, 288). Typically, two transgenes areinserted into a vector between a promoter and transcription terminatoras two separate open reading frames separated by an IRES. Upon deliveryof the constructs to a host cell, a single long transcript encoding bothtransgenes will be transcribed. The first ORF will be translated in thetraditional cap-dependent manner, terminating at a stop codon upstreamof the IRES. The second ORF will be translated in a cap-independentmanner using the IRES. In this way, two independent proteins can beproduced from a single mRNA transcribed from a vector with a singleexpression cassette.

Although the multi-antigen expression strategies are described here inthe context of a DNA vaccine construct, the principles apply similarlyin the context of viral vector genetic vaccines.

D. Vectors Containing a Nucleic Acid Molecule Encoding an ImmunogenicPAA Polypeptide

Another aspect of the invention relates to vectors containing one ormore nucleic acid molecules of the invention. The vectors are useful forcloning or expressing the immunogenic PAA polypeptides encoded by thenucleic acid molecules, or for delivering the nucleic acid molecule in acomposition, such as a vaccine, to a host cell or to a host animal, suchas a human. A wide variety of vectors may be prepared to contain andexpress a nucleic acid molecule of the invention, such as plasmidvectors, cosmid vectors, phage vectors, and viral vectors.

In some embodiments, the disclosure provides a plasmid-based vectorcontaining a nucleic acid molecule of the invention. Representativeexamples of suitable plasmid vectors include pBR325, pUC18, pSKF,pET23D, and pGB-2. Other representative examples of plasmid vectors, aswell as method of constructing such vectors, are described in U.S. Pat.Nos. 5,580,859, 5,589,466, 5,688,688, 5,814,482 and 5,580,859.

In other embodiments, the present invention provides vectors that areconstructed from viruses, such as retroviruses, alphaviruses,adenoviruses. Representative examples of retroviral vectors aredescribed in more detail in EP 0,415,731; PCT Publication Nos. WO90/07936; WO 91/0285, WO 9311230; WO 9310218, WO 9403622; WO 9325698; WO9325234; and U.S. Pat. Nos. 5,219,740, 5,716,613, 5,851,529, 5,591,624,5,716,826, 5,716,832, and 5,817,491. Representative examples of vectorsthat can be generated from alphaviruses are described in U.S. Pat. Nos.5,091,309 and 5,217,879, 5,843,723, and 5,789,245. In some particularembodiments, the present disclosure provides adenoviral vectors derivedfrom non-human primate adenoviruses, such as simian adenoviruses.Examples of such adenoviral vectors, as well as their preparation, aredescribed in PCT application publication WO2005/071093 and WO2010/086189, and include non-replicating vectors such as ChAd3, ChAd4,ChAd5, ChAd7, ChAd8, ChAd9, ChAd10, ChAd11, ChAd16, ChAd17, ChAd19,ChAd20, ChAd22, ChAd24, ChAd26, ChAd30, ChAd31, ChAd37, ChAd38, ChAd44,ChAd63, ChAd68, ChAd82, ChAd55, ChAd73, ChAd83, ChAd146, ChAd147,PanAd1, Pan Ad2, and Pan Ad3, and replication-competent vectors such asAd4 and Ad7 vectors. It is preferred that in constructing the adenoviralvectors from the simian adenoviruses one or more of the early genes fromthe genomic region of the virus selected from E1A, E1B, E2A, E2B, E3,and E4 are either deleted or rendered non-functional by deletion ormutation. In a particular embodiment, the vector is constructed fromChAd3 or ChAd68. Suitable vectors can also be generated from otherviruses such as: pox viruses, such as canary pox virus or vaccinia virus(Fisher-Hoch et al., PNAS 86:317-321, 1989; Flexner et al., Ann. N.Y.Acad. Sci. 569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S.Pat. Nos. 4,603,112, 4,769,330 and 5,017,487; WO 89/01973);adeno-associated vectors (see, e.g., U.S. Pat. No. 5,872,005); SV40(Mulligan et al., Nature 277:108-114, 1979); herpes (Kit, Adv. Exp. Med.Biol. 215:219-236, 1989; U.S. Pat. No. 5,288,641); and lentivirus suchas HIV (Poznansky, J. Virol. 65:532-536, 1991).

Methods of constructing vectors are well known in the art. Expressionvectors typically include one or more control elements that areoperatively linked to the nucleic acid sequence to be expressed. Theterm “control elements” refers collectively to promoter regions,polyadenylation signals, transcription termination sequences, upstreamregulatory domains, origins of replication, internal ribosome entrysites (“IRES”), enhancers, and the like, which collectively provide forthe replication, transcription, and translation of a coding sequence ina recipient cell. Not all of these control elements need always bepresent so long as the selected coding sequence is capable of beingreplicated, transcribed, and translated in an appropriate host cell. Thecontrol elements are selected based on a number of factors known tothose skilled in that art, such as the specific host cells and source orstructures of other vector components, For enhancing the expression ofan immunogenic PAA polypeptide, a Kozak sequence can be providedupstream of the sequence encoding the immunogenic PAA polypeptide. Forvertebrates, a known Kozak sequence is (GCC)NCCATGG, wherein N is A or Gand GCC is less conserved. Exemplary Kozak sequences that can be usedinclude ACCAUGG and ACCATGG.

E. Compositions Comprising an Immunogenic PAA Polypeptide (PolypeptideCompositions)

In another aspect, the present disclosure provides compositionscomprising one or more isolated immunogenic PAA polypeptides provided bythe present disclosure (“polypeptide composition”). In some embodiments,the polypeptide composition is an immunogenic composition useful foreliciting an immune response against a PAA protein in a mammal, such asa mouse, dog, nonhuman primates or human. In some other embodiments, thepolypeptide composition is a vaccine composition useful for immunizationof a mammal, such as a human, for inhibiting abnormal cellproliferation, for providing protection against the development ofcancer (used as a prophylactic), or for treatment of disorders (used asa therapeutic) associated with PAA over expression, such as cancers,particularly prostate cancer.

A polypeptide composition provided by the present disclosure may containa single type of immunogenic PAA polypeptide, such an immunogenic PSMApolypeptide, an immunogenic PSA polypeptide, or an immunogenic PSCApolypeptide. A composition may also contain a combination of two or moredifferent types of immunogenic PAA polypeptides. For example, apolypeptide composition may contain immunogenic PAA polypeptides in anyof the following combinations:

1) an immunogenic PSMA polypeptide and an immunogenic PSA polypeptide;

2) an immunogenic PSMA polypeptide and a PSCA polypeptide; or

3) an immunogenic PSMA polypeptide, an immunogenic PSA polypeptide, anda PSCA polypeptide.

An immunogenic composition or vaccine composition provided by thepresent disclosure may further comprise a pharmaceutically acceptableexcipient. Pharmaceutically acceptable excipients for immunogenic orvaccine compositions are known in the art. Examples of suitableexcipients include biocompatible oils, such as rape seed oil, sunfloweroil, peanut oil, cotton seed oil, jojoba oil, squalan, squalene,physiological saline solution, preservatives and osmotic pressurecontrolling agents, carrier gases, pH-controlling agents, organicsolvents, hydrophobic agents, enzyme inhibitors, water absorbingpolymers, surfactants, absorption promoters, pH modifiers, andanti-oxidative agents.

The immunogenic PAA polypeptide in a composition, particularly animmunogenic composition or a vaccine composition, may be linked to,conjugated to, or otherwise incorporated into a carrier foradministration to a recipient. The term “carrier” refers to a substanceor structure that an immunogenic polypeptide can be attached to orotherwise associated with for delivery of the immunogenic polypeptide tothe recipient (e.g., patient). The carrier itself may be immunogenic.Examples of carriers include immunogenic polypeptides, immune CpGislands, limpet hemocyanin (KLH), tetanus toxoid (TT), cholera toxinsubunit B (CTB), bacteria or bacterial ghosts, liposome, chitosome,virosomes, microspheres, dendritic cells, or their like. One or moreimmunogenic PAA polypeptide molecules may be linked to a single carriermolecule. Methods for linking an immunogenic polypeptide to a carrierare known in the art,

A vaccine composition or immunogenic composition provided by the presentdisclosure may be used in conjunction with one or more immune modulatorsor adjuvants. The immune modulators or adjuvants may be formulatedseparately from the vaccine composition, or they may be part of the samevaccine composition formulation. Thus, in one embodiment, the vaccinecomposition further comprises one or more immune modulators oradjuvants. Examples of immune modulators and adjuvants are providedherein below.

The polypeptide compositions, including the immunogenic and vaccinecompositions, can be prepared in any suitable dosage forms, such asliquid forms (e.g., solutions, suspensions, or emulsions) and solidforms (e.g., capsules, tablets, or powder), and by methods known to oneskilled in the art.

F. Compositions Comprising an Immunogenic PAA Nucleic Acid Molecule(Nucleic Acid Compositions)

The present disclosure also provides a composition comprising anisolated nucleic acid molecule or vector provided by the presentdisclosure (herein “nucleic acid composition’). The nucleic acidcompositions are useful for eliciting an immune response against a PAAprotein in vitro or in vivo in a mammal, including a human.

In some particular embodiments, the nucleic acid composition is a DNAvaccine composition for administration to humans for inhibiting abnormalcell proliferation, providing protection against the development ofcancer (used as a prophylactic), or for treatment of cancer (used as atherapeutic) associated with PAA over-expression, or for eliciting animmune response to a particular human PAA, such as PSMA, PSA, and PSCA.The nucleic acid molecule in the composition may be a “naked” nucleicacid molecule, i.e. simply in the form of an isolated DNA free fromelements that promote transfection or expression. Alternatively, thenucleic acid molecule in the composition can be incorporated into avector.

A nucleic acid composition provided by the present disclosure maycomprise individual isolated nucleic acid molecules that each encodeonly one type of immunogenic PAA polypeptide, such as an immunogenicPSMA polypeptide, an immunogenic PSA polypeptide, or an immunogenic PSCApolypeptide.

A nucleic acid composition may comprise a multi-antigen constructprovided by the present disclosure that encodes two or more types ofimmunogenic PAA polypeptides. A multi-antigen construct may encode twoor more immunogenic PAA polypeptides in any of the followingcombinations:

1) an immunogenic PSMA polypeptide and an immunogenic PSA polypeptide;

2) an immunogenic PSMA polypeptide and an immunogenic PSCA polypeptide;

3) an immunogenic PSA polypeptide and an immunogenic PSCA polypeptide;and

4) an immunogenic PSMA polypeptide, an immunogenic PSA polypeptide, andan immunogenic PSCA polypeptide.

The nucleic acid compositions, including the DNA vaccine compositions,may further comprise a pharmaceutically acceptable excipient. Examplesof suitable pharmaceutically acceptable excipients for nucleic acidcompositions, including DNA vaccine compositions, are well known tothose skilled in the art and include sugars, etc. Such excipients may beaqueous or non aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous excipients include propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Examples of aqueous excipient include water,alcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media. Suitable excipients also include agents that assistin cellular uptake of the polynucleotide molecule. Examples of suchagents are (i) chemicals that modify cellular permeability, such asbupivacaine, (ii) liposomes or viral particles for encapsulation of thepolynucleotide, or (iii) cationic lipids or silica, gold, or tungstenmicroparticles which associate themselves with the polynucleotides.Anionic and neutral liposomes are well-known in the art (see, e.g.,Liposomes: A Practical Approach, RPC New Ed, IRL press (1990), for adetailed description of methods for making liposomes) and are useful fordelivering a large range of products, including polynucleotides.Cationic lipids are also known in the art and are commonly used for genedelivery. Such lipids include Lipofectin™ also known as DOTMA(N—[I-(2,3-dioleyloxy) propyls N,N, N-trimethylammonium chloride), DOTAP(1,2-bis (oleyloxy)-3 (trimethylammonio) propane), DDAB(dimethyldioctadecyl-ammonium bromide), DOGS (dioctadecylamidologlycylspermine) and cholesterol derivatives such as DCChol (3beta-(N—(N′,N′-dimethyl aminomethane)-carbamoyl) cholesterol). Adescription of these cationic lipids can be found in EP 187,702, WO90/11092, U.S. Pat. No. 5,283,185, WO 91/15501, WO 95/26356, and U.S.Pat. No. 5,527,928. A particular useful cationic lipid formulation thatmay be used with the nucleic vaccine provided by the disclosure isVAXFECTIN, which is a commixture of a cationic lipid (GAP-DMORIE) and aneutral phospholipid (DPyPE) which, when combined in an aqueous vehicle,self-assemble to form liposomes. Cationic lipids for gene delivery arepreferably used in association with a neutral lipid such as DOPE(dioleyl phosphatidylethanolamine), as described in WO 90/11092 as anexample. In addition, a DNA vaccine can also be formulated with anonionic block copolymer such as CRL1005.

G. Uses of the Immunogenic PAA Polypeptides, Nucleic Acid Molecules, andCompositions

In other aspects, the present disclosure provides methods of using theimmunogenic PAA polypeptides, isolated nucleic acid molecules, andcompositions comprising an immunogenic PAA polypeptide or isolatednucleic acid molecule described herein above.

In one aspect, the present disclosure provides a method of eliciting animmune response against a PAA in a mammal, particularly a human,comprising administering to the mammal an effective amount of (1) animmunogenic PAA polypeptide provided by the disclosure that isimmunogenic against the target PAA, (2) an isolated nucleic acidmolecule encoding such an immunogenic PAA polypeptide, (3) a compositioncomprising such an immunogenic PAA polypeptide, or (4) a compositioncomprising an isolated nucleic acid molecule encoding such animmunogenic PAA polypeptide.

In some embodiments, the disclosure provides a method of eliciting animmune response against PSMA in a human, comprising administering to thehuman an effective amount of an immunogenic PSMA composition provided bythe present disclosure, wherein the immunogenic PSMA composition isselected from: (1) an immunogenic PSMA polypeptide, (2) an isolatednucleic acid molecule encoding an immunogenic PSMA polypeptide, (3) acomposition comprising an immunogenic PSMA polypeptide, or (4) acomposition comprising an isolated nucleic acid molecule encoding animmunogenic PSMA polypeptide.

In some other embodiments, the disclosure provides a method of elicitingan immune response against PSA in a human, comprising administering tothe human an effective amount of an immunogenic PSA composition providedby the present disclosure, wherein the immunogenic PSA composition isselected from: (1) an immunogenic PSA polypeptide, (2) an isolatednucleic acid molecule encoding an immunogenic PSA polypeptide, (3) acomposition comprising an immunogenic PSA polypeptide, or (4) acomposition comprising an isolated nucleic acid molecule encoding animmunogenic PSA polypeptide.

In another aspect, the present disclosure provides a method ofinhibiting abnormal cell proliferation in a human, wherein the abnormalcell proliferation is associated with over-expression of a PAA. Themethod comprises administering to the human an effective amount ofimmunogenic PAA composition provided by the present disclosure that isimmunogenic against the over-expressed PAA. The immunogenic PAAcomposition may be (1) an immunogenic PAA polypeptide, (2) an isolatednucleic acid molecule encoding one or more immunogenic PAA polypeptides,(3) a composition comprising an immunogenic PAA polypeptide, or (4) acomposition comprising an isolated nucleic acid molecule encoding one ormore immunogenic PAA polypeptides. In some embodiments, the method isfor inhibiting abnormal cell proliferation in prostate in a human. In aparticular embodiment, the present disclosure provide a method ofinhibiting abnormal cell proliferation in prostate over-expressing PSMA,comprising administering to the human effective amount of (1) animmunogenic PSMA polypeptide, (2) an isolated nucleic acid moleculeencoding one or more immunogenic PSMA polypeptides, (3) a compositioncomprising an immunogenic PSMA polypeptide, or (4) a compositioncomprising an isolated nucleic acid molecule encoding one or moreimmunogenic PSMA polypeptide.

In another aspect, the present disclosure provides a method of treatingcancer in a human wherein cancer is associated with over-expression of aPAA. The method comprises administering to the human an effective amountof immunogenic PAA composition capable of eliciting an immune responseagainst the over-expressed PAA. The immunogenic PAA composition may be(1) an immunogenic PAA polypeptide, (2) an isolated nucleic acidmolecule encoding one or more immunogenic PAA polypeptides, (3) acomposition comprising an immunogenic PAA polypeptide, or (4) acomposition comprising an isolated nucleic acid molecule encoding one ormore immunogenic PAA polypeptides. Examples of cancers that may betreated with the method include breast cancer, stomach cancer, ovariancancer, lung cancer, bladder cancer, colorectal cancer, renal cancer,pancreatic cancer and prostate cancer.

In some embodiments, the disclosure provides a method of treatingprostate cancer in a human, comprising administering to the human aneffective amount of a nucleic acid composition provided herein above.The nucleic acids in the composition may encode only one particularimmunogenic PAA polypeptide, such an immunogenic PSMA polypeptide, animmunogenic PSA polypeptide, or an immunogenic PSCA polypeptide. Thenucleic acids in the composition may also encode two or more differentimmunogenic PAA polypeptides, such as: (1) an immunogenic PSMApolypeptide and an immunogenic PSA polypeptide; (2) an immunogenic PSMApolypeptide and an immunogenic PSCA polypeptide; (3) an immunogenic PSApolypeptide and an immunogenic PSCA polypeptide; (4) an immunogenic PSMApolypeptide, an immunogenic PSA polypeptide, and an immunogenic PSCApolypeptide. Each individual nucleic acid molecule in the compositionmay encode only one particular immunogenic PAA polypeptide, such as aPSMA polypeptide, a PSA polypeptide, or a PSCA polypeptide.Alternatively, an individual nucleic acid molecule in the compositionmay be a multi-antigen constructs encoding two different types ofimmunogenic PAA polypeptides, such as: (1) an immunogenic PSMApolypeptide and an immunogenic PSA polypeptide; (2) an immunogenic PSMApolypeptide and an immunogenic PSCA polypeptide; (3) an immunogenic PSCApolypeptide and an immunogenic PSA polypeptide; or (4) an immunogenicPSMA polypeptide, an immunogenic PSA polypeptide, and an immunogenicPSCA polypeptide. In some particular embodiments, the nucleic acidcomposition comprises a multi-antigen construct that encode at least (4)an immunogenic PSMA polypeptide, an immunogenic PSA polypeptide, and animmunogenic PSCA polypeptide. The immunogenic PSCA polypeptide containedin vaccine compositions or expressed by a nucleic acid in vaccinecompositions for the treatment of prostate cancer in human is preferablythe human full length PSCA protein.

The polypeptide and nucleic acid compositions can be administered to ananimal, including human, by a number of methods known in the art.Examples of suitable methods include: (1) intramuscular, intradermal,intraepidermal, intravenous, intraarterial, subcutaneous, orintraperitoneal administration, (2) oral administration, and (3) topicalapplication (such as ocular, intranasal, and intravaginal application).One particular method of intradermal or intraepidermal administration ofa nucleic acid vaccine composition that may be used is gene gun deliveryusing the Particle Mediated Epidermal Delivery (PMED™) vaccine deliverydevice marketed by PowderMed. PMED is a needle-free method ofadministering vaccines to animals or humans. The PMED system involvesthe precipitation of DNA onto microscopic gold particles that are thenpropelled by helium gas into the epidermis. The DNA-coated goldparticles are delivered to the APCs and keratinocytes of the epidermis,and once inside the nuclei of these cells, the DNA elutes off the goldand becomes transcriptionally active, producing encoded protein. Thisprotein is then presented by the APCs to the lymphocytes to induce aT-cell-mediated immune response. Another particular method forintramuscular administration of a nucleic acid vaccine provided by thepresent disclosure is electroporation. Electroporation uses controlledelectrical pulses to create temporary pores in the cell membrane, whichfacilitates cellular uptake of the nucleic acid vaccine injected intothe muscle. Where a CpG is used in combination with a nucleic acidvaccine, it is preferred that the CpG and nucleic acid vaccine areco-formulated in one formulation and the formulation is administeredintramuscularly by electroporation.

The effective amount of the immunogenic PAA polypeptide or nucleic acidencoding an immunogenic PAA polypeptide in the composition to beadministered in a given method provided by the present disclosure can bereadily determined by a person skilled in the art and will depend on anumber of factors. In a method of treating cancer, such as prostatecancer, factors that may be considered in determining the effectiveamount of the immunogenic PAA polypeptide or nucleic acid include, butnot limited: (1) the subject to be treated, including the subject'simmune status and health, (2) the severity or stage of the cancer to betreated, (3) the specific immunogenic PAA polypeptides used orexpressed, (4) the degree of protection or treatment desired, (5) theadministration method and schedule, and (6) other therapeutic agents(such as adjuvants or immune modulators) used. In the case of nucleicacid vaccine compositions, including the multi-antigen vaccinecompositions, the method of formulation and delivery are among the keyfactors for determining the dose of the nucleic acid required to elicitan effective immune response. For example, the effective amounts of thenucleic acid may be in the range of 2 μg/dose-10 mg/dose when thenucleic acid vaccine composition is formulated as an aqueous solutionand administered by hypodermic needle injection or pneumatic injection,whereas only 16 ng/dose-16 μg/dose may be required when the nucleic acidis prepared as coated gold beads and delivered using a gene guntechnology. The dose range for a nucleic acid vaccine by electroporationis generally in the range of 0.5-10 mg/dose. In the case where thenucleic acid vaccine is administered together with a CpG byelectroporation in a co-formulation, the dose of the nucleic acidvaccine may be in the range of 0.5-5 mg/dose and the dose of CpG istypically in the range of 0.05 mg-5 mg/dose, such as 0.05, 0.2, 0.6, or1.2 mg/dose per person.

The nucleic acid or polypeptide vaccine composition of the presentinvention can be used in a prime-boost strategy to induce robust andlong-lasting immune response. Priming and boosting vaccination protocolsbased on repeated injections of the same immunogenic construct are wellknown. In general, the first dose may not produce protective immunity,but only “primes” the immune system. A protective immune responsedevelops after the second or third dose (the “boosts). The boosts areperformed according to conventional techniques, and can be furtheroptimized empirically in terms of schedule of administration, route ofadministration, choice of adjuvant, dose, and potential sequence whenadministered with another vaccine. In one embodiment, the nucleic acidor polypeptide vaccines of the present invention are used in aconventional homologous prime-boost strategy, in which the same vaccineis administered to the animal in multiple doses. In another embodiment,the nucleic acid or polypeptide vaccine compositions are used in aheterologous prime-boost vaccination, in which different types ofvaccines containing the same antigens are administered at predeterminedtime intervals. For example, a nucleic acid construct may beadministered in the form of a plasmid in the initial dose (“prime”) andas part of a vector in the subsequent doses (“boosts”), or vice versa.

For the treatment of prostate cancer, the polypeptide or nucleic acidvaccines of the present invention may be used together with prostatecancer vaccines based on other antigens, such as prostatic acidphosphatase-based antigens and androgen receptor.

The polypeptide or nucleic acid vaccine composition of the presentinvention may be used together with one or more adjuvants. Examples ofsuitable adjuvants include: (1) oil-in-water emulsion formulations (withor without other specific immunostimulating agents such as muramylpolypeptides or bacterial cell wall components), such as for example (a)MF59™ (PCT Publication No. WO 90/14837; Chapter 10 in Vaccine design:the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press1995), containing 5% Squalene, 0.5% Tween 80 (polyoxyethylene sorbitanmono-oleate), and 0.5% Span 85 (sorbitan trioleate) formulated intosubmicron particles using a microfluidizer, (b) SAF, containing 10%Squalene, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDPeither microfluidized into a submicron emulsion or vortexed to generatea larger particle size emulsion, and (c) RIBI™ adjuvant system (RAS)(Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween80, and one or more bacterial cell wall components such asmonophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wallskeleton (CWS); (2) saponin adjuvants, such as QS21, STIMULON™(Cambridge Bioscience, Worcester, Mass.), Abisco® (Isconova, Sweden), orIscomatrix® (Commonwealth Serum Laboratories, Australia); (3) CompleteFreund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (4)cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6,IL-7, IL-12 (PCT Publication No. WO 99/44636), etc.), interferons (e.g.gamma interferon), macrophage colony stimulating factor (M-CSF), tumornecrosis factor (TNF), etc.; (5) monophosphoryl lipid A (MPL) or3-O-deacylated MPL (3dMPL), optionally in the substantial absence ofalum when used with pneumococcal saccharides (e.g. GB-2220221,EP-A-0689454, WO 00/56358); (6) combinations of 3dMPL with, for example,QS21 and/or oil-in-water emulsions (e.g. EP-A-0835318, EP-A-0735898,EP-A-0761231); (7) oligonucleotides comprising CpG motifs, i.e.containing at least one CG dinucleotide, where the cytosine isunmethylated (e.g., Krieg, Vaccine (2000) 19:618-622; Krieg, Curr OpinMol Ther (2001) 3:15-24; WO 98/40100, WO 98/55495, WO 98/37919 and WO98/52581); (8) a polyoxyethylene ether or a polyoxyethylene ester (e.g.WO 99/52549); (9) a polyoxyethylene sorbitan ester surfactant incombination with an octoxynol (e.g., WO 01/21207) or a polyoxyethylenealkyl ether or ester surfactant in combination with at least oneadditional non-ionic surfactant such as an octoxynol (e.g., WO01/21152); (10) a saponin and an immunostimulatory oligonucleotide (e.g.a CpG oligonucleotide) (e.g., WO 00/62800); (11) metal salt includingaluminum salts (such as alum, aluminum phosphate, aluminum hydroxide);(12) a saponin and an oil-in-water emulsion (e.g. WO 99/11241); (13) asaponin (e.g. QS21)+3dMPL+IM2 (optionally+a sterol)(e.g. WO 98/57659);(14) other substances that act as immunostimulating agents to enhancethe efficacy of the composition, such as Muramyl polypeptides includingN-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutarninyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE), (15) ligands for toll-like receptors (TLR), natural orsynthesized (e.g. Kanzler et al., Nature Med. 13:1552-1559 (2007)),including TLR3 ligands such as polyl:C and similar compounds such asHiltonol and Ampligen.

The polypeptide or nucleic acid vaccine compositions of the presentinvention may be used together with one or more immune modulators.Examples of suitable immune modulators include protein tyrosine kinaseinhibitors (such as afatinib, axitinib, cediranib, erlotinib, gefitinib,grandinin, lapatinib, lestaurtinib, neratinib, pazopanib, quizartinib,regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib,bosutinib and vandetanib), CD40 agonists (such as CD40 agonistantibody), OX40 agonists (such as OX40 agonist antibody), CTLA-4inhibitors (such as antiCTLA-4 antibody Ipilimumab and Tremelimumab),TLR agonists, 4-1BB agonists, Tim-1 antagonists, LAGE-3 antagonists andPD-L1 & PD-1 antagonists.

H. Vaccine-Based Immunotherapy Regimens (VBIR)

In a further aspect, the present disclosure provides a method ofenhancing the immunogenicity or therapeutic effect of a vaccine for thetreatment of a neoplastic disorder in a mammal, particularly a human.The method comprises administering to the mammal receiving the vaccinefor the treatment of a neoplastic disorder (1) an effective amount of atleast one immune-suppressive-cell inhibitor (ISC inhibitor) and (2) aneffective amount of at least one immune-effector-cell enhancer (IECenhancer). The method may be used in combination with a vaccine in anyform or formulation, for example, a subunit vaccine, a protein-basedvaccine, a peptide-based vaccine, or a nucleic acid-based vaccines suchas a DNA-based vaccine, a RNA-based vaccine, a plasmid-based vaccine, ora vector-based vaccine. In addition, the method is not limited to anyparticular types of vaccines or any particular types of cancer. Rather,the method may be used in combination with any vaccine intended for thetreatment of neoplastic disorder, including benign, pre-malignant, andmalignant neoplastic disorders. For example, the method may be used incombination a vaccine that is intended for the treatment of any of thefollowing neoplastic disorders: carcinoma including that of the bladder(including accelerated and metastatic bladder cancer), breast, colon(including colorectal cancer), kidney, liver, lung (including small andnon-small cell lung cancer and lung adenocarcinoma), ovary, prostate,testes, genitourinary tract, lymphatic system, rectum, larynx, pancreas(including exocrine pancreatic carcinoma), esophagus, stomach, gallbladder, cervix, thyroid, and skin (including squamous cell carcinoma);hematopoietic tumors of lymphoid lineage including leukemia, acutelymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma,T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy celllymphoma, histiocytic lymphoma, and Burketts lymphoma; hematopoietictumors of myeloid lineage including acute and chronic myelogenousleukemias, myelodysplastic syndrome, myeloid leukemia, and promyelocyticleukemia; tumors of the central and peripheral nervous system includingastrocytoma, neuroblastoma, glioma, and schwannomas; tumors ofmesenchymal origin including fibrosarcoma, rhabdomyosarcoma, andosteosarcoma; other tumors including melanoma, xenoderma pigmentosum,keratoactanthoma, seminoma, thyroid follicular cancer, andteratocarcinoma; melanoma, unresectable stage III or IV malignantmelanoma, squamous cell carcinoma, small-cell lung cancer, non-smallcell lung cancer, glioma, gastrointestinal cancer, renal cancer, ovariancancer, liver cancer, colorectal cancer, endometrial cancer, kidneycancer, prostate cancer, thyroid cancer, neuroblastoma, pancreaticcancer, glioblastoma multiforme, cervical cancer, stomach cancer,bladder cancer, hepatoma, breast cancer, colon carcinoma, and head andneck cancer, gastric cancer, germ cell tumor, bone cancer, bone tumors,adult malignant fibrous histiocytoma of bone; childhood malignantfibrous histiocytoma of bone, sarcoma, pediatric sarcoma, sinonasalnatural killer, neoplasms, plasma cell neoplasm; myelodysplasticsyndromes; neuroblastoma; testicular germ cell tumor, intraocularmelanoma, myelodysplastic syndromes; myelodysplastic/myeloproliferativediseases, synovial sarcoma, chronic myeloid leukemia, acutelymphoblastic leukemia, philadelphia chromosome positive acutelymphoblastic leukemia (Ph+ALL), multiple myeloma, acute myelogenousleukemia, chronic lymphocytic leukemia, and mastocytosis.

In some embodiments, present disclosure provides a method of enhancingthe immunogenicity or therapeutic effect of a vaccine for the treatmentof prostate cancer in a human. The vaccine administered may be capableof eliciting an immune response against any human PAA, such as PSMA,PSA, or PSCA. In some particular embodiments, the vaccine administeredcomprises a nucleic acid molecule encoding an antigen capable ofeliciting immunogenicity against a human PAA, such as PSMA, PSA, orPSCA. Examples of specific nucleic acid molecules that may be containedin the vaccine include the following provided by the present disclosure:

1) a nucleic acid molecule encoding an immunogenic PSMA polypeptide, animmunogenic PSA polypeptide, or an immunogenic PSCA polypeptide;

2) a nucleic acid molecule encoding two immunogenic PAA polypeptidesprovided by the present disclosure, such as a) an immunogenic PSMApolypeptide and an immunogenic PSA polypeptide; b) an immunogenic PSMApolypeptide and an immunogenic PSCA polypeptide; or c) an immunogenicPSA polypeptide and an immunogenic PSCA polypeptide; and

3) a nucleic acid molecule encoding three immunogenic PAA polypeptides,which are an immunogenic PSMA polypeptide, an immunogenic PSApolypeptide, and an immunogenic PSCA polypeptide.

In another further aspect, the present disclosure provides a method oftreating a neoplastic disorder in a mammal, particularly a human. Themethod comprises administering to the mammal (1) an effective amount ofa vaccine capable of eliciting an immune response against a TAAassociated with the neoplastic disorder, (2) an effective amount of atleast one immune-suppressive-cell inhibitor (ISC inhibitor), and (3) aneffective amount of at least one immune-effector-cell enhancer (IECenhancer). Any vaccine that is capable of eliciting an immune responseagainst a particular TAA may be used in the method. Many TAAs are knownin the art. In addition to the prostate-associated antigens, thefollowing are examples of TAAs that are known in the art: CEA, MUC-1,Ep-CAM, 5T4, hCG-b, K-ras, and TERT for colorectal cancer; CEA, Muc-1,p53, mesothelin, Survivin, and NY-ESO-1 for ovarian cancer; Muc-1, 5T4,WT-1, TERT, CEA, EGF-R and MAGE-A3 for non-small cell lung cancer; 5T4for renal cell carcinoma; and Muc-1, mesothelin, K-Ras, Annexin A2,TERT, and CEA for pancreatic cancer. New TAAs continue to be identified.A vaccine that is capable of eliciting an immune response against any ofthe known or new TAAs can be used in the method. In addition, thevaccine administered may be in any form or formulation, for example,subunit vaccines, protein-based vaccine, peptide based vaccines, ornucleic acid-based vaccines such DNA-based vaccines, RNA-based vaccines,plasmid-based vaccines, or vector-based vaccines.

In some embodiments, the present disclosure provides a method oftreating a prostate cancer in a human, the method comprisingadministering to the human a vaccine capable of eliciting an immuneresponse against any human PAA, such as PSMA, PSA, or PSCA. In someparticular embodiments, the vaccine administered comprises a nucleicacid molecule encoding an antigen capable of eliciting immunogenicityagainst a human PAA, such as PSMA, PSA, or PSCA. Examples of specificnucleic acid molecules that may be contained in the vaccine include thefollowing provided by the present disclosure:

1) a nucleic acid molecule encoding an immunogenic PSMA polypeptide, animmunogenic PSA polypeptide, or an immunogenic PSCA polypeptide;

2) a nucleic acid molecule encoding two immunogenic PAA polypeptidesprovided by the present disclosure, such as a) an immunogenic PSMApolypeptide and an immunogenic PSA polypeptide; b) an immunogenic PSMApolypeptide and an immunogenic PSCA polypeptide; or c) an immunogenicPSA polypeptide and an immunogenic PSCA polypeptide; and

3) a nucleic acid molecule encoding three immunogenic PAA polypeptides,which are an immunogenic PSMA polypeptide, an immunogenic PSApolypeptide, and an immunogenic PSMA polypeptide.

The method of treating a neoplastic disorder in a mammal and the methodof enhancing the immunogenicity or therapeutic effect of a vaccine forthe treatment of a neoplastic disorder in a mammal described hereinabove are alternatively referred to as “vaccine-based immunotherapyregimens” (or “VBIR”).

In the vaccine-based immunotherapy regimens, the IEC enhancers and ISCinhibitors may be administered by any suitable methods and routes,including (1) systemic administration such as intravenous,intramuscular, or oral administration, and (2) local administration suchintradermal and subcutaneous administration. Where appropriate orsuitable, local administration is generally preferred over systemicadministration. Local administration of any IEC enhancer and ISCinhibitor can be carried out at any location of the body of the mammalthat is suitable for local administration of pharmaceuticals; however,it is more preferable that these immune modulators are administeredlocally at close proximity to the vaccine draining lymph node.

Two or more specific IEC enhancers from a single class of IEC enhancers(for examples, two CTLA-agonists) may be administered in combinationwith the ISC inhibitors. In addition, two or more specific IEC enhancersfrom two or more different classes of IEC enhancers (for example, oneCTLA-4 antagonist and one TLR agonist) may be administered together.Similarly, two or more specific ISC inhibitors from a single class ofISC inhibitors (for examples, two or more protein kinase inhibitors) maybe administered in combination with the IEC enhancers. In addition, twoor more specific ISC inhibitors from two or more different classes ofISC inhibitors (for example, one protein kinase inhibitor and one COX-2inhibitor) may be administered together.

In the vaccine-based immunotherapy regimens the vaccine may beadministered simultaneously or sequentially with any or all of theimmune modulators (i.e., ISC inhibitors and IEC enhancers) used.Similarly, when two or more immune modulators are used, they may beadministered simultaneously or sequentially with respect to each other.In some embodiments, a vaccine is administered simultaneously (e.g., ina mixture) with respect to one immune modulator, but sequentially withrespect to one or more additional immune modulators. Co-administrationof the vaccine and the immune modulators in the vaccine-basedimmunotherapy regimen can include cases in which the vaccine and atleast one immune modulator are administered so that each is present atthe administration site, such as vaccine draining lymph node, at thesame time, even though the antigen and the immune modulators are notadministered simultaneously. Co-administration of the vaccine and theimmune modulators also can include cases in which the vaccine or theimmune modulator is cleared from the administration site, but at leastone cellular effect of the cleared vaccine or immune modulator persistsat the administration site, such as vaccine draining lymph node, atleast until one or more additional immune modulators are administered tothe administration site. In cases where a nucleic acid vaccine isadministered in combination with a CpG, the vaccine and CpG may becontained in a single formulation and administered together by anysuitable method. In some embodiments, the nucleic acid vaccine and CpGin a co-formulation (mixture) is administered by intramuscular injectionin combination with electroporation.

Any ISC inhibitors may be used in the vaccine-based immunotherapyregimens. Examples of classes of SIC inhibitors include protein kinaseinhibitors, cyclooxygenase-2 (COX-2) inhibitors, phosphodiesterase type5 (PDE5) inhibitors, and DNA crosslinkers. Examples COX-2 inhibitorsinclude celecoxib and rofecoxib. Examples of PDE5 inhibitors includeavanafil, lodenafil, mirodenafil, sildenafil, tadalafil, vardenafil,udenafil, and zaprinast. An example of DNA crosslinkers iscyclophosphamide. Examples of specific protein kinase inhibitors aredescribed in details below.

The term “protein kinase inhibitor” refers to any substance that acts asa selective or non-selective inhibitor of a protein kinase. The term“protein kinases” refers to the enzymes that catalyze the transfer ofthe terminal phosphate of adenosine triphosphate to tyrosine, serine orthreonine residues in protein substrates. Protein kinases includereceptor tyrosine kinases and non-receptor tyrosine kinases. Examples ofreceptor tyrosine kinases include EGFR (e.g., EGFR/HER1/ErbB1,HER2/Neu/ErbB2, HER3/ErbB3, HER4/ErbB4), INSR (insulin receptor),IGF-IR, IGF-II1R, IRR (insulin receptor-related receptor), PDGFR (e.g.,PDGFRA, PDGFRB), c-KIT/SCFR, VEGFR-1/FLT-1, VEGFR-2/FLK-1/KDR,VEGFR-3/FLT-4, FLT-3/FLK-2, CSF-1R, FGFR 1-4, CCK4, TRK A-C, MET, RON,EPHA 1-8, EPHB 1-6, AXL, MER, TYRO3, TIE, TEK, RYK, DDR 1-2, RET, c-ROS,LTK (leukocyte tyrosine kinase), ALK (anaplastic lymphoma kinase), ROR1-2, MUSK, AATYK 1-3, and RTK 106. Examples of non-receptor tyrosinekinases include BCR-ABL, Src, Frk, Btk, Csk, Abl, Zap70, Fes/Fps, Fak,Jak, Ack, and LIMK. In the vaccine-based immunotherapy regimen providedby the present disclosure, the protein kinase inhibitors areadministered to the mammal at a suboptimal dose. The term “suboptimaldose” refers to the dose amount that is below the minimum effective dosewhen the tyrosine kinase inhibitor is administered in a monotherapy(i.e., where the protein kinase inhibitor is administered alone withoutany other therapeutic agents) for the target neoplastic disorder.

Examples of specific protein kinase inhibitors suitable for use in thevaccine-based immunotherapy regimen include Lapatinib, AZD 2171, ETI8OCH3, Indirubin-3′-oxime, NSC-154020, PD 169316, Quercetin, Roscovitine,Triciribine, ZD 1839, 5-lodotubercidin, Adaphostin, Aloisine,Alsterpaullone, Aminogenistein, API-2, Apigenin, Arctigenin,ARRY-334543, Axitinib (AG-013736), AY-22989, AZD 2171,Bisindolylmaleimide IX, CCI-779, Chelerythrine, DMPQ, DRB, Edelfosine,ENMD-981693, Erbstatin analog, Erlotinib, Fasudil, Gefitinib (ZD1839),H-7, H-8, H-89, HA-100, HA-1004, HA-1077, HA-1100, Hydroxyfasudil,Kenpaullone, KN-62, KY12420, LFM-A13, Luteolin, LY294002, LY-294002,Mallotoxin, ML-9, MLN608, NSC-226080, NSC-231634, NSC-664704,NSC-680410, NU6102, Olomoucine, Oxindole I, PD 153035, PD 98059,Phloridzin, Piceatannol, Picropodophyllin, PKI, PP1, PP2,PTK787/ZK222584, PTK787/ZK-222584, Purvalanol A, Rapamune, Rapamycin, Ro31-8220, Rottlerin, SB202190, SB203580, Sirolimus, SL327, SP600125,Staurosporine, STI-571, SU1498, SU4312, SU5416, SU5416 (Semaxanib),SU6656, SU6668, syk inhibitor, TBB, TCN, Tyrphostin AG 1024, TyrphostinAG 490, Tyrphostin AG 825, Tyrphostin AG 957, U0126, W-7, Wortmannin,Y-27632, Zactima (ZD6474), ZM 252868. gefitinib (Iressa®), sunitinibmalate (Sutent®; SU11248), erlotinib (Tarceva®; OSI-1774), lapatinib(GW572016; GW2016), canertinib (CI 1033), semaxinib (SU5416), vatalanib(PTK787/ZK222584), sorafenib (BAY 43-9006), imatinib (Gleevec®; STI571),dasatinib (BMS-354825), leflunomide (SU101), vandetanib (Zactima®;ZD6474), and nilotinib. Additional protein kinase inhibitors suitablefor use in the present invention are described in, e.g., U.S. Pat. Nos.5,618,829, 5,639,757, 5,728,868, 5,804,396, 6,100,254, 6,127,374,6,245,759, 6,306,874, 6,313,138, 6,316,444, 6,329,380, 6,344,459,6,420,382, 6,479,512, 6,498,165, 6,544,988, 6,562,818, 6,586,423,6,586,424, 6,740,665, 6,794,393, 6,875,767, 6,927,293, and 6,958,340.

In some embodiments, the protein kinase inhibitor is a multi-kinaseinhibitor, which is an inhibitor that acts on more than one specifickinase. Examples of multi-kinase inhibitors include imatinib, sorafenib,lapatinib, BIRB-796, and AZD-1152, AMG706, Zactima (ZD6474), MP-412,sorafenib (BAY 43-9006), dasatinib, CEP-701 (lestaurtinib), XL647,XL999, Tykerb (lapatinib), MLN518, (formerly known as CT53518), PKC412,ST1571, AEE 788, OSI-930, OSI-817, sunitinib malate (Sutent), axitinib(AG-013736), erlotinib, gefitinib, axitinib, bosutinib, temsirolismusand nilotinib (AMN107). In some particular embodiments, the tyrosinekinase inhibitor is sunitinib, sorafenib, or a pharmaceuticallyacceptable salt or derivative (such as a malate or a tosylate) ofsunitinib or sorafenib.

Sunitinib malate, which is marketed by Pfizer Inc. under the trade nameSUTENT, is described chemically as butanedioic acid, hydroxy-, (2S)-,compound withN-[2-(diethylamino)ethyl]-5-[(Z)-(5-fluoro-1,2-dihydro-2-oxo-3H-indol-3-ylidine)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide(1:1). The compound, its synthesis, and particular polymorphs aredescribed in U.S. Pat. No. 6,573,293, U.S. Patent Publication Nos.2003-0229229, 2003-0069298 and 2005-0059824, and in J. M. Manley, M. J.Kalman, B. G. Conway, C. C. Ball, J. L Havens and R. Vaidyanathan,“Early Amidation Approach to 3-[(4-amido)pyrrol-2-yl]-2-indolinones,” J.Org. Chew. 68, 6447-6450 (2003). Formulations of sunitinib and itsL-malate salt are described in PCT Publication No. WO 2004/024127.Sunitinib malate has been approved in the U.S. for the treatment ofgastrointestinal stromal tumor, advanced renal cell carcinoma, andprogressive, well-differentiated pancreatic neuroendocrine tumors inpatients with unresectable locally advanced or metastatic disease. Therecommended dose of sunitinib malate for gastrointestinal stromal tumor(GIST) and advanced renal cell carcinoma (RCC) for humans is 50 mg takenorally once daily, on a schedule of 4 weeks on treatment followed by 2weeks off (Schedule 4/2). The recommended dose of sunitinib malate forpancreatic neuroendocrine tumors (pNET) is 37.5 mg taken orally oncedaily.

In the vaccine-based immunotherapy regimen, sunitinib malate may beadministered orally in a single dose or multiple doses. Typically,sunitinib malate is delivered for two, three, four or more consecutiveweekly doses followed by a “off” period of about 1 or 2 weeks, or morewhere no sunitinib malate is delivered. In one embodiment, the doses aredelivered for about 4 weeks, with 2 weeks off. In another embodiment,the sunitinib malate is delivered for two weeks, with 1 week off.However, it may also be delivered without a “off’ period for the entiretreatment period. The effective amount of sunitinib malate administeredorally to a human in the vaccine-based immunotherapy regimen istypically below 40 mg per person per dose. For example, it may beadministered orally at 37.5, 31.25, 25, 18.75, 12.5, 6.25 mg per personper day. In some embodiments, sunitinib malate is administered orally inthe range of 1-25 mg per person per dose. In some other embodiments,sunitinib malate is administered orally in the range of 6.25, 12.5, or18.75 mg per person per dose. Other dosage regimens and variations areforeseeable, and will be determined through physician guidance.

Sorafenib tosylate, which is marketed under the trade name NEXAVAR, isalso a multi-kinase inhibitor. Its chemical name is4-(4-{3-[4-Chloro-3-(trifluoromethyl)phenyl]ureido}phenoxy)-N-methylpyrid-ine-2-carboxamide.It is approved in the U.S. for the treatment of primary kidney cancer(advanced renal cell carcinoma) and advanced primary liver cancer(hepatocellular carcinoma). The recommended daily dose is 400 mg takenorally twice daily. In the vaccine-based immunotherapy regimen providedby the present disclosure, the effective amount of sorafenib tosylateadministered orally is typically below 400 mg per person per day. Insome embodiments, the effective amount of sorafenib tosylateadministered orally is in the range of 10-300 mg per person per day. Insome other embodiments, the effective amount of sorafenib tosylateadministered orally is between 10-200 mg per person per day, such as 10,20, 60, 80, 100, 120, 140, 160, 180, or 200 mg per person per day.

Axitinib, which is marketed under the trade name INLYTA, is a selectiveinhibitor of VEGF receptors 1, 2, and 3. Its chemical name is(N-Methyl-2-[3-((E)-2-pyridin-2-yl-vinyl)-1H-indazol-6-ylsulfany]-benzamide.It is approved for the treatment of advanced renal cell carcinoma afterfailure of one prior systemic therapy. The starting dose is 5 mg orallytwice daily. Dose adjustments can be made based on individual safety andtolerability. In the vaccine-based immunotherapy regimen provided by thepresent disclosure, the effective amount of axitinib administered orallyis typically below 5 mg twice daily. In some other embodiments, theeffective amount of axitinib administered orally is between 1-5 mg twicedaily. In some other embodiments, the effective amount of axitinibadministered orally is between 1, 2, 3, 4, or 5 mg twice daily.

In the vaccine-based immunotherapy regimens any IEC enhancers may beused. They may be small molecules or large molecules (such as protein,polypeptide, DNA, RNA, and antibody). Examples of IEC enhancers that maybe used include TNFR agonists, CTLA-4 antagonists, TLR agonists,programmed cell death protein 1 (PD-1) antagonists (such as BMS-936558),anti-PD-1 antibody CT-011), and programmed cell death protein 1 ligand 1(PD-L1) antagonists (such as BMS-936559), lymphocyte-activation gene 3(LAG3) antagonists, and T cell Immunoglobulin- andmucin-domain-containing molecule-3 (TIM-3) antagonists. Examples ofspecific TNFR agonists, CTLA-4 antagonists, and TLR agonists areprovided in details herein below.

TNFR Agonists.

Examples of TNFR agonists include agonists of OX40, 4-1BB (such asBMS-663513), GITR (such as TRX518), and CD40. Examples of specific CD40agonists are described in details herein below.

CD40 agonists are substances that bind to a CD40 receptor on a cell andis capable of increasing one or more CD40 or CD40L associatedactivities. Thus, CD40 “agonists” encompass CD40 “ligands”.

Examples of CD40 agonists include CD40 agonistic antibodies, fragmentsCD40 agonistic antibodies, CD40 ligands (CD40L), and fragments andderivatives of CD40L such as oligomeric (e.g., bivalent, trimericCD40L), fusion proteins containing and variants thereof.

CD40 ligands for use in the present invention include any peptide,polypeptide or protein, or a nucleic acid encoding a peptide,polypeptide or protein that can bind to and activate one or more CD40receptors on a cell. Suitable CD40 ligands are described, for example,in U.S. Pat. Nos. 6,482,411, 6,410,711; U.S. Pat. No. 6,391,637; andU.S. Pat. No. 5,981,724, all of which patents and application and theCD40L sequences disclosed therein are incorporated by reference in theirentirety herein. Although human CD40 ligands will be preferred for usein human therapy, CD40 ligands from any species may be used in theinvention. For use in other animal species, such as in veterinaryembodiments, a species of CD40 ligand matched to the animal beingtreated will be preferred. In certain embodiments, the CD40 ligand is agp39 peptide or protein oligomer, including naturally forming gp39peptide, polypeptide or protein oligomers, as well as gp39 peptides,polypeptides, proteins (and encoding nucleic acids) that comprise anoligomerization sequence. While oligomers such as dimers, trimers andtetramers are preferred in certain aspects of the invention, in otheraspects of the invention larger oligomeric structures are contemplatedfor use, so long as the oligomeric structure retains the ability to bindto and activate one or more CD40 receptor(s).

In certain other embodiments, the CD40 agonist is an anti-CD40 antibody,or antigen-binding fragment thereof. The antibody can be a human,humanized or part-human chimeric anti-CD40 antibody. Examples ofspecific anti-CD40 monoclonal antibodies include the G28-5, mAb89, EA-5or S2C6 monoclonal antibody, and CP870893. In a particular embodiment,the anti-CD40 agonist antibody is CP870893 or dacetuzumab (SGN-40).

CP-870,893 is a fully human agonistic CD40 monoclonal antibody (mAb)that has been investigated clinically as an anti-tumor therapy. Thestructure and preparation of CP870,893 is disclosed in WO2003041070(where the antibody is identified by the internal identified “21.4.1”).The amino acid sequences of the heavy chain and light chain ofCP-870,893 are set forth in SEQ ID NO: 40 and SEQ ID NO: 41,respectively. In clinical trials, CP870,893 was administered byintravenous infusion at doses generally in the ranges of 0.05-0.25 mg/kgper infusion. In a phase I clinical study, the maximum tolerated dose(MTD) of CP-870893 was estimated to be 0.2 mg/kg and the dose-limitingtoxicities included grade 3 CRS and grade 3 urticaria. [Jens Ruter etal.: Immune modulation with weekly dosing of an agonist CD40 antibody ina phase I study of patients with advanced solid tumors. Cancer Biology &Therapy 10:10, 983-993; Nov. 15, 2010.]. In the vaccine-basedimmunotherapy regimen provided by the present disclosure, CP-870,893 canbe administered intradermally, subcutaneously, or topically. It ispreferred that it is administered intradermally. The effective amount ofCP870893 to be administered in the regimen is generally below 0.2 mg/kg,typically in the range of 0.01 mg-0.15 mg/kg, or 0.05-0.1 mg/kg.

Dacetuzumab (also known as SGN-40 or huS2C6; CAS number 88-486-59-9) isanother anti-CD40 agonist antibody that has been investigated inclinical trials for indolent lymphomas, diffuse large B cell lymphomasand Multiple Myeloma. In the clinical trials, dacetuzumab wasadministered intravenously at weekly doses ranging from 2 mg/kg to 16mg/kg. In the vaccine-based immunotherapy regimen provided by thepresent disclosure, dacetuzumab can be administered intradermally,subcutaneously, or topically. It is preferred that it is administeredintradermally. The effective amount of dacetuzumab to be administered inthe vaccine-based immunotherapy regimen is generally below 16 mg/kg,typically in the range of 0.2 mg-14 mg/kg, or 0.5-8 mg/kg, or 1-5 mg/kg.

CTLA-4 Inhibitors.

Suitable anti-CTLA-4 antagonist agents for use in the vaccine-basedimmunotherapy regimen provided by the disclosure include, withoutlimitation, anti-CTLA-4 antibodies (such as human anti-CTLA-4antibodies, mouse anti-CTLA-4 antibodies, mammalian anti-CTLA-4antibodies, humanized anti-CTLA-4 antibodies, monoclonal anti-CTLA-4antibodies, polyclonal anti-CTLA-4 antibodies, chimeric anti-CTLA-4antibodies, anti-CTLA-4 domain antibodies), fragments of anti-CTLA-4antibodies (such as (single chain anti-CTLA-4 fragments, heavy chainanti-CTLA-4 fragments, and light chain anti-CTLA-4 fragments), andinhibitors of CTLA-4 that agonize the co-stimulatory pathway. In someembodiments, the CTLA-4 inhibitor is Ipilimumab or Tremelimumab.

Ipilimumab (also known as MEX-010 or MDX-101), marketed as YERVOY, is ahuman anti-human CTLA-4 antibody. Ipilimumab can also be referred to byits CAS Registry No. 477202-00-9, and is disclosed as antibody 10D1 inPCT Publication No. WO 01/14424, incorporated herein by reference in itsentirety and for all purposes. Examples of pharmaceutical compositioncomprising Ipilimumab are provided in PCT Publication No. WO 2007/67959.Ipilimumab is approved in the U.S. for the treatment of unresectable ormetastatic melanoma. The recommended dose of Ipilimumab as monotherapyis 3 mg/kg by intravenous administration every 3 weeks for a total of 4doses. In the methods provided by the present invention, Ipilimumab isadministered locally, particularly intradermally or subcutaneously. Theeffective amount of Ipilimumab administered locally is typically in therange of 5-200 mg/dose per person. In some embodiments, the effectiveamount of Ipilimumab is in the range of 10-150 mg/dose per person perdose. In some particular embodiments, the effective amount of Ipilimumabis about 10, 25, 50, 75, 100, 125, 150, 175, or 200 mg/dose per person.

Tremelimumab (also known as CP-675,206) is a fully human IgG2 monoclonalantibody and has the CAS number 745013-59-6. Tremelimumab is disclosedas antibody 11.2.1 in U.S. Pat. No. 6,682,736, incorporated herein byreference in its entirety and for all purposes. The amino acid sequencesof the heavy chain and light chain of Tremelimumab are set forth in SEQIND NOs:42 and 43, respectively. Tremelimumab has been investigated inclinical trials for the treatment of various tumors, including melanomaand breast cancer; in which Tremelimumab was administered intravenouslyeither as single dose or multiple doses every 4 or 12 weeks at the doserange of 0.01 and 15 mg/kg. In the regimens provided by the presentinvention, Tremelimumab is administered locally, particularlyintradermally or subcutaneously. The effective amount of Tremelimumabadministered intradermally or subcutaneously is typically in the rangeof 5-200 mg/dose per person. In some embodiments, the effective amountof Tremelimumab is in the range of 10-150 mg/dose per person per dose.In some particular embodiments, the effective amount of Tremelimumab isabout 10, 25, 50, 75, 100, 125, 150, 175, or 200 mg/dose per person.

Toll-Like Receptor (TLR) Agonists.

The term “toll-like receptor agonist” or “TLR agonist” refers to acompound that acts as an agonist of a toll-like receptor (TLR). Thisincludes agonists of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLR10, and TLR11 or a combination thereof. Unless otherwiseindicated, reference to a TLR agonist compound can include the compoundin any pharmaceutically acceptable form, including any isomer (e.g.,diastereomer or enantiomer), salt, solvate, polymorph, and the like. Inparticular, if a compound is optically active, reference to the compoundcan include each of the compound's enantiomers as well as racemicmixtures of the enantiomers. Also, a compound may be identified as anagonist of one or more particular TLRs (e.g., a TLR7 agonist, a TLR8agonist, or a TLR7/8 agonist).

The TLR agonism for a particular compound may be assessed in anysuitable manner known in the art. Regardless of the particular assayemployed, a compound can be identified as an agonist of a particular TLRif performing the assay with a compound results in at least a thresholdincrease of some biological activity mediated by the particular TLR.Conversely, a compound may be identified as not acting as an agonist ofa specified TLR if, when used to perform an assay designed to detectbiological activity mediated by the specified TLR, the compound fails toelicit a threshold increase in the biological activity. Unless otherwiseindicated, an increase in biological activity refers to an increase inthe same biological activity over that observed in an appropriatecontrol. An assay may or may not be performed in conjunction with theappropriate control. With experience, one skilled in the art may developsufficient familiarity with a particular assay (e.g., the range ofvalues observed in an appropriate control under specific assayconditions) that performing a control may not always be necessary todetermine the TLR agonism of a compound in a particular assay.

Certain TLR agonists useful in the method of the present invention aresmall organic molecules, as opposed to large biological molecules suchas proteins, peptides, and the like. Examples of small molecule TLRagonists include those disclosed in, for example, U.S. Pat. Nos.4,689,338; 4,929,624; 4,988,815; 5,037,986; 5,175,296; 5,238,944;5,266,575; 5,268,376; 5,346,905; 5,352,784; 5,367,076; 5,389,640;5,395,937; 5,446,153; 5,482,936; 5,693,811; 5,741,908; 5,756,747;5,939,090; 6,039,969; 6,083,505; 6,110,929; 6,194,425; 6,245,776;6,331,539; 6,376,669; 6,451,810; 6,525,064; 6,545,016; 6,545,017;6,558,951; and 6,573,273. Examples of specific small molecule TLRagonists useful in the methods provided by the present invention include4-amino-alpha, alpha,2-trimethyl-IH-imidazo[4,5-c]qumolin-l-ethanol,N-(2-{2-[4-amino-2-(2-methoxyethyl)-IH-imidazo[4,5-c]quinolin-I-yl]ethoxy-}ethyl)-N-methylmorpholine-4-carboxamide,I˜(2˜amino-2-methylpropyl)-2-(ethoxymethyl-)-IH-imidazo[4,5-c]quinolin-4-arnine,N-[4-(4-amino-2-ethyl-IH-imidazo[4,5-c]quinolin-I-yl)butyl]methanesulfonamide,N-[4-(4-amino-2-propyl-IH-imidazo[4,5-c]quinolin-I-yl)butyl]me-thanesulfonamide,and imiquimod. Some TLR agonists particularly useful in the methods orregimen provided by the present disclosure are discussed in reviewarticle: Folkert Steinhagen, et al.: TLR-based immune adjuvants. Vaccine29 (2011): 3341-3355.

In some embodiments, the TLR agonists are TLR9 agonists, particularlyCpG oligonucleotides (or CpG.ODN). A CpG oligonucleotide is a shortnucleic acid molecule containing a cytosine followed by a guanine linkedby a phosphate bond in which the pyrimidine ring of the cytosine isunmethylated. A CpG motif is a pattern of bases that include anunmethylated central CpG surrounded by at least one base flanking (onthe 3′ and the 5′ side of) the central CpG. CpG oligonucleotides includeboth D and K oligonucleotides. The entire CpG oligonucleotide can beunmethylated or portions may be unmethylated. Examples of CpGoligonucleotides useful in the methods provided by the presentdisclosure include those disclosed in U.S. Pat. Nos. 6,194,388,6,207,646, 6,214,806, 628,371, 6,239,116, and 6,339,068.

The CpG oligonucleotides can encompass various chemical modificationsand substitutions, in comparison to natural RNA and DNA, involving aphosphodiester internucleoside bridge, a beta-D-ribose (deoxyhbose) unitand/or a natural nucleoside base (adenine, guanine, cytosine, thymine,uracil). Examples of chemical modifications are known to the skilledperson and are described, for example in Uhlmann E. et al. (1990), Chem.Rev. 90:543; “Protocols for Oligonucleotides and Analogs”, Synthesis andProperties and Synthesis and Analytical Techniques, S. Agrawal, Ed.,Humana Press, Totowa, USA 1993; Crooke, S T. et al. (1996) Annu. Rev.Pharmacol. Toxicol. 36:107-129; and Hunziker J. et al., (1995), Mod.Synth. Methods 7:331-417. Specifically, a CpG oligonucleotide cancontain a modified cytosine. A modified cytosine is a naturallyoccurring or non-naturally occurring pyrimidine base analog of cytosinewhich can replace this base without impairing the immunostimulatoryactivity of the oligonucleotide. Modified cytosines include but are notlimited to 5-substituted cytosines (e.g. 5-methyl-cytosine,5-fluorocytosine, 5-chloro-cytosine, 5-bromo-cytosine, 5-iodo-cytosine,5-hydroxy-cytosine, 5-hydroxymethyl-cytosine, 5-difluoromethyl-cytosine,and unsubstituted or substituted 5-alkynyl-cytosine), 6-substitutedcytosines, N4-substituted cytosines (e.g. N4-ethyl-cytosine),5-aza-cytosine, 2-mercapto-cytosine, isocytosine, pseudo-isocytosine,cytosine analogs with condensed ring systems (e.g. N,N′-propylenecytosine or phenoxazine), and uracil and its derivatives (e.g.5-fluoro-uracil, 5-bromo-uracil, 5-bromovinyl-uracil, 4-thio-uracil,5-hydroxy-uracil, 5-propynyl-uracil). Some of the preferred cytosinesinclude 5-methyl-cytosine, 5-fluoro-cytosine, 5-hydroxy-cytosine,5-hydroxymethyl-cytosine, and N4-ethyl-cytosine.

A CpG oligonucleotide can also contain a modified guanine. A modifiedguanine is a naturally occurring or non-naturally occurring purine baseanalog of guanine which can replace this base without impairing theimmunostimulatory activity of the oligonucleotide. Modified guaninesinclude but are not limited to 7-deeazaguanine, 7-deaza-7-substitutedguanine, hypoxanthine, N2-substituted guanines (e.g. N2-methyl-guanine),5-amino-3-methyl-3H,6H-thiazolo[4,5-d]pyhmidine-2,7-dione,2,6-diaminopuhne, 2-aminopuhne, purine, indole, adenine, substitutedadenines (e.g. N6-methyl-adenine, 8-oxo-adenine), 8-substituted guanine(e.g. 8-hydroxyguanine and 8-bromoguanine), and 6-thioguanine. In someembodiments of the disclosure, the guanine base is substituted by auniversal base (e.g. 4-methyl-indole, 5-nitro-indole, and K-base), anaromatic ring system (e.g. benzimidazole or dichloro-benzimidazole,1-methyl-1H-[1,2,4]triazole-3-carboxylic acid amide) or a hydrogen atom.

In certain aspects, the CpG oligonucleotides include modified backbones.It has been demonstrated that modification of the nucleic acid backboneprovides enhanced activity of nucleic acids when administered in vivo.Secondary structures, such as stem loops, can stabilize nucleic acidsagainst degradation. Alternatively, nucleic acid stabilization can beaccomplished via phosphate backbone modifications. A preferredstabilized nucleic acid has at least a partial phosphorothioate modifiedbackbone. Phosphorothioates may be synthesized using automatedtechniques employing either phosphoramidate or H-phosphonatechemistries. Aryl- and alkyl-phosphonates can be made, e.g. as describedin U.S. Pat. No. 4,469,863; and alkylphosphotriesters (in which thecharged oxygen moiety is alkylated as described in U.S. Pat. No.5,023,243 and European Patent No. 092,574) can be prepared by automatedsolid phase synthesis using commercially available reagents. Methods formaking other DNA backbone modifications and substitutions have beendescribed (Uhlmann, E. and Peyman, A. (1990) Chem. Rev. 90:544;Goodchild, J. (1990) Bioconjugate Chem. 1:165). 2′-O-methyl nucleicacids with CpG motifs also cause immune activation, as doethoxy-modified CpG nucleic acids. In fact, no backbone modificationshave been found that completely abolish the CpG effect, although it isgreatly reduced by replacing the C with a 5-methyl C. Constructs havingphosphorothioate linkages provide maximal activity and protect thenucleic acid from degradation by intracellular exo- and endo-nucleases.Other modified oligonucleotides include phosphodiester modifiedoligonucleotides, combinations of phosphodiester and phosphorothioateoligonucleotides, methylphosphonate, methylphosphorothioate,phosphorordithioate, p-ethoxy, and combinations thereof. Each of thesecombinations and their particular effects on immune cells is discussedin more detail with respect to CpG nucleic acids in PCT Publication Nos.WO 96/02555 and WO 98/18810 and in U.S. Pat. Nos. 6,194,388 and6,239,116.

The CpG oligonucleotides may have one or two accessible 5′ ends. It ispossible to create modified oligonucleotides having two such 5′ ends,for instance, by attaching two oligonucleotides through a 3′-3′ linkageto generate an oligonucleotide having one or two accessible 5′ ends. The3′-3′-linkage may be a phosphodiester, phosphorothioate or any othermodified internucleoside bridge. Methods for accomplishing such linkagesare known in the art. For instance, such linkages have been described inSeliger, H. et al., Oligonucleotide analogs with terminal 3′-3′- and5′-5′-internucleotidic linkages as antisense inhibitors of viral geneexpression, Nucleosides and Nucleotides (1991), 10(1-3), 469-77 andJiang, et al., Pseudo-cyclic oligonucleotides: in vitro and in vivoproperties, Bioorganic and Medicinal Chemistry (1999), 7(12), 2727-2735.

Additionally, 3′-3′-linked oligonucleotides where the linkage betweenthe 3′-terminal nucleosides is not a phosphodiester, phosphorothioate orother modified bridge, can be prepared using an additional spacer, suchas tri- or tetra-ethyleneglycol phosphate moiety (Durand, M. et al.,Triple-helix formation by an oligonucleotide containing one (dA)12 andtwo (dT)12 sequences bridged by two hexaethylene glycol chains,Biochemistry (1992), 31 (38), 9197-204, U.S. Pat. Nos. 5,658,738 and5,668,265). Alternatively, the non-nucleotidic linker may be derivedfrom ethanediol, propanediol, or from an abasic deoxyhbose (dSpacer)unit (Fontanel, Marie Laurence et al., Nucleic Acids Research (1994),22(11), 2022-7) using standard phosphoramidite chemistry. Thenon-nucleotidic linkers can be incorporated once or multiple times, orcombined with each other allowing for any desirable distance between the3′-ends of the two oligonucleotides to be linked.

A phosphodiester internucleoside bridge located at the 3′ and/or the 5′end of a nucleoside can be replaced by a modified internucleosidebridge, wherein the modified internucleoside bridge is for exampleselected from phosphorothioate, phosphorodithioate,NRiR₂-phosphoramidate, boranophosphate, a-hydroxybenzyl phosphonate,phosphate-(C₁-C₂₁)—O-alkyl ester,phosphate-[(C₆-C₂₁)aryl-(C₁-C₂₁)—O-alkyl]ester, (C1-C₈)alkylphosphonateand/or (C₆—C1₂)arylphosphonate bridges, (C₇-C₁₂)-a-hydroxymethyl-aryl(e.g. disclosed in PCT Publication No. WO 95/01363), wherein(C₆-C₁₂)aryl, (C₆-C₂₀)aryl and (C₆-C₁₄)aryl are optionally substitutedby halogen, alkyl, alkoxy, nitro, cyano, and where Ri and R₂ are,independently of each other, hydrogen, (C₁-C1₈)-alkyl, (C₆-C₂₀)-aryl,(C₆-C₁₄)-aryl, (C₁-C₈)-alkyl, preferably hydrogen, (C₁-C₈)-alkyl,preferably (C₁-C₄)-alkyl and/or methoxyethyl, or R₁ and R₂ form,together with the nitrogen atom carrying them, a 5 to 6-memberedheterocyclic ring which can additionally contain a further heteroatomselected from the group O, S and N.

The replacement of a phosphodiester bridge located at the 3′ and/or the5′ end of a nucleoside by a dephospho bridge (dephospho bridges aredescribed, for example, in Uhlmann E. and Peyman A. in “Methods inMolecular Biology”, Vol. 20, “Protocols for Oligonucleotides andAnalogs”, S. Agrawal, Ed., Humana Press, Totowa 1993, Chapter 16, pp.355 if), wherein a dephospho bridge is for example selected from thedephospho bridges formacetal, 3′-thioformacetal, methylhydroxylamine,oxime, methylenedimethyl-hydrazo, dimethylenesulfone and/or silylgroups.

The CpG oligonucleotides for use in the methods or regimen provided bythe disclosure may optionally have chimeric backbones. A chimericbackbone is one that comprises more than one type of linkage. In oneembodiment, the chimeric backbone can be represented by the formula: 5′Y1 N1ZN2Y2 3′. Y1 and Y2 are nucleic acid molecules having between 1 and10 nucleotides. Y1 and Y2 each include at least one modifiedinternucleotide linkage. Since at least 2 nucleotides of the chimericoligonucleotides include backbone modifications these nucleic acids arean example of one type of “stabilized immunostimulatory nucleic acids”.

With respect to the chimeric oligonucleotides, Y1 and Y2 are consideredindependent of one another. This means that each of Y1 and Y2 may or maynot have different sequences and different backbone linkages from oneanother in the same molecule. In some embodiments, Y1 and/or Y2 havebetween 3 and 8 nucleotides. N1 and N2 are nucleic acid molecules havingbetween 0 and 5 nucleotides as long as N1ZN2 has at least 6 nucleotidesin total. The nucleotides of N1ZN2 have a phosphodiester backbone and donot include nucleic acids having a modified backbone. Z is animmunostimulatory nucleic acid motif, preferably selected from thoserecited herein.

The center nucleotides (N1ZN2) of the formula Y1 N1ZN2Y2 havephosphodiester internucleotide linkages and Y1 and Y2 have at least one,but may have more than one or even may have all modified internucleotidelinkages. In preferred embodiments, Y1 and/or Y2 have at least two orbetween two and five modified internucleotide linkages or Y1 has fivemodified internucleotide linkages and Y2 has two modifiedinternucleotide linkages. The modified internucleotide linkage, in someembodiments, is a phosphorothioate modified linkage, aphosphorodithioate linkage or a p-ethoxy modified linkage.

Examples of particular CpG oligonucleotides useful in the methodsprovided by the present disclosure include:

5′ TCGTCGTTTTGTCGTTTTGTCGTT3′; (CpG 7909) 5′ TCGTCGTTTTTCGGTGCTTTT3′;(CpG 24555) and 5′ TCGTCGTTTTTCGGTCGTTTT3′. (CpG 10103)

CpG7909, a synthetic 24mer single stranded, has been extensivelyinvestigated for the treatment of cancer as a monotherapy and incombination with chemotherapeutic agents, as well as adjuvant as anadjuvant for vaccines against cancer and infectious diseases. It wasreported that a single intravenous dose of CpG 7909 was well toleratedwith no clinical effects and no significant toxicity up to 1.05 mg/kg,while a single dose subcutaneous CpG 7909 had a maximum tolerated dose(MTD) of 0.45 mg/kg with dose limiting toxicity of myalgia andconstitutional effects. [See Zent, Clive S, et al: Phase I clinicaltrial of CpG oligonucleotide 7909 (PF-03512676) in patients withpreviously treated chronic lymphocytic leukemia. Leukemia and Lymphoma,53(2):211-217(7)(2012). In the regimens provided by the presentdisclosure, CpG7909 may be administered by injection into the muscle orany other suitable methods. It is preferred that it is administeredlocally in proximity to the vaccine draining lymph node, particularly byintradermal or subcutaneous administration. For use with a nucleic acidvaccine, such as a DNA vaccine, a CpG may be preferably co-formulatedwith the vaccine in a single formulation and administered byintramuscular injection coupled with electroporation. The effectiveamount of CpG7909 by intramuscular, intradermal, or subcutaneousadministration is typically in the range of 10 μg/dose-10 mg/dose. Insome embodiments, the effective amount of CpG7909 is in the range of0.05 mg-14 mg/dose. In some particular embodiments, the effective amountof CpG7909 is about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 05 1 mg/dose. OtherCpG oligonucleotides, including CpG 24555 and CpG 10103, may beadministered in similar manner and dose levels.

In some particular embodiments, the present disclosure provides a methodof enhancing the immunogenicity or therapeutic effect of a vaccine forthe treatment of a neoplastic disorder in a human, comprisingadministering the human (1) an effective amount of at least one ISCinhibitor and (2) an effective amount of at least one IEC enhancer,wherein the at least one ISC inhibitor is protein kinase inhibitorselected from sorafenib tosylate, sunitinib malate, axitinib, erlotinib,gefitinib, axitinib, bosutinib, temsirolismus, or nilotinib and whereinthe at least one IEC enhancer is selected from a CTLA-4 inhibitor, a TLRagonist, or a CD40 agonist. In some preferred embodiments, regimencomprises administering to the human (1) an effective amount of at leastone ISC inhibitor and (2) effective amount of at least one IEC enhancer,wherein the at least one ISC inhibitor is a protein kinase inhibitorselected from axitinib, sorafenib tosylate, or sunitinib malate andwherein the wherein the at least one IEC enhancer is a CTLA-4 inhibitorselected from Ipilimumab or Tremelimumab. In some further preferredembodiments, the regimen comprises administering to the human (1) aneffective amount of at least one ISC inhibitor and (2) an effectiveamount of at least two IEC enhancers, wherein the at least one ISCinhibitor is a protein kinase inhibitor selected from sunitinib oraxitinib and wherein the at least two IEC enhancers are Tremelimumab anda TLR agonist selected from CpG7909, CpG2455, or CpG 10103.

In some other embodiments, the present disclosure provides a method oftreating prostate cancer in a human, comprising administering to thehuman (1) an effective amount of a vaccine capable of eliciting animmune response against a human PAA, (2) an effective amount of at leastone ISC inhibitor, and (3) an effective amount of at least one IECenhancer, wherein the at least one ISC inhibitor is a protein kinaseinhibitor selected from sorafenib tosylate, sunitinib malate, axitinib,erlotinib, gefitinib, axitinib, bosutinib, temsirolismus, or nilotinib,and wherein the at least one IEC enhancer is selected from a CTLA-4inhibitor, a TLR agonist, or a CD40 agonist. In some preferredembodiments, the method comprises administering to the human (1) aneffective amount of a vaccine capable of eliciting an immune responseagainst a human PAA, (2) an effective amount of at least one ISCinhibitor, and (3) an effective amount of at least one IEC enhancer,wherein the at least one ISC inhibitor is a protein kinase inhibitorselected from sorafenib tosylate, sunitinib malate, or axitinib andwherein the at least one IEC enhancer is a CTLA-4 inhibitor selectedfrom Ipilimumab or Tremelimumab.

In some further specific embodiments, the method comprises administeringto the human (1) an effective amount of at least one ISC inhibitor and(2) an effective amount of at least two IEC enhancers, wherein the atleast one ISC inhibitor is a protein kinase inhibitor selected fromsunitinib or axitinib and wherein the at least two IEC enhancers areTremelimumab and a TLR agonist selected from CpG7909, CpG2455, orCpG10103.

Additional Therapeutic Agents.

The vaccine-based immunotherapy regimen provided by the presentdisclosure may further comprise an additional therapeutic agent. A widevariety of cancer therapeutic agents may be used, includingchemotherapeutic agents and hormone therapeutic agents. One of ordinaryskill in the art will recognize the presence and development of othercancer therapies which can be used in VBIR provided by the presentdisclosure, and will not be restricted to those forms of therapy setforth herein.

The term “chemotherapeutic agent” refers to a chemical or biologicalsubstance that can cause death of cancer cells, or interfere withgrowth, division, repair, and/or function of cancer cells. Examples ofchemotherapeutic agents include those that are disclosed inWO2006/088639, WO2006/129163, and US 20060153808, the disclosures ofwhich are incorporated herein by reference. Examples of particularchemotherapeutic agents include: (1) alkylating agents, such aschlorambucil (LEUKERAN), cyclophosphamide (CYTOXAN), ifosfamide (IFEX),mechlorethamine hydrochloride (MUSTARGEN), thiotepa (THIOPLEX),streptozotocin (ZANOSAR), carmustine (BICNU, GLIADEL WAFER), lomustine(CEENU), and dacarbazine (DTIC-DOME); (2) alkaloids or plant vincaalkaloids, including cytotoxic antibiotics, such as doxorubicin(ADRIAMYCIN), epirubicin (ELLENCE, PHARMORUBICIN), daunorubicin(CERUBIDINE, DAUNOXOME), nemorubicin, idarubicin (IDAMYCIN PFS,ZAVEDOS), mitoxantrone (DHAD, NOVANTRONE). dactinomycin (actinomycin D,COSMEGEN), plicamycin (MITHRACIN), mitomycin (MUTAMYCIN), and bleomycin(BLENOXANE), vinorelbine tartrate (NAVELBINE)), vinblastine (VELBAN),vincristine (ONCOVIN), and vindesine (ELDISINE); (3) antimetabolites,such as capecitabine (XELODA), cytarabine (CYTOSAR-U), fludarabine(FLUDARA), gemcitabine (GEMZAR), hydroxyurea (HYDRA), methotrexate(FOLEX, MEXATE, TREXALL), nelarabine (ARRANON), trimetrexate(NEUTREXIN), and pemetrexed (ALIMTA); (4) Pyrimidine antagonists, suchas 5-fluorouracil (5-FU); capecitabine (XELODA), raltitrexed (TOMUDEX),tegafur-uracil (UFTORAL), and gemcitabine (GEMZAR); (5) taxanes, such asdocetaxel (TAXOTERE), paclitaxel (TAXOL); (6) platinum drugs, such ascisplatin (PLATINOL) and carboplatin (PARAPLATIN), and oxaliplatin(ELOXATIN); (7) topoisomerase inhibitors, such as irinotecan(CAMPTOSAR), topotecan (HYCAMTIN), etoposide (ETOPOPHOS, VEPESSID,TOPOSAR), and teniposide (VUMON); (8) epipodophyllotoxins(podophyllotoxin derivatives), such as etoposide (ETOPOPHOS, VEPESSID,TOPOSAR); (9) folic acid derivatives, such as leucovorin (WELLCOVORIN);(10) nitrosoureas, such as carmustine (BiCNU), lomustine (CeeNU); (11)inhibitors of receptor tyrosine kinase, including epidermal growthfactor receptor (EGFR), vascular endothelial growth factor (VEGF),insulin receptor, insulin-like growth factor receptor (IGFR), hepatocytegrowth factor receptor (HGFR), and platelet-derived growth factorreceptor (PDGFR), such as gefitinib (IRESSA), erlotinib (TARCEVA),bortezomib (VELCADE), imatinib mesylate (GLEEVEC), genefitinib,lapatinib, sorafenib, thalidomide, sunitinib (SUTENT), axitinib,rituximab (RITUXAN, MABTHERA), trastuzumab (HERCEPTIN), cetuximab(ERBITUX), bevacizumab (AVASTIN), and ranibizumab (LUCENTIS), lym-1(ONCOLYM), antibodies to insulin-like growth factor-1 receptor (IGF-1R)that are disclosed in WO2002/053596); (12) angiogenesis inhibitors, suchas bevacizumab (AVASTIN), suramin (GERMANIN), angiostatin, SU5416,thalidomide, and matrix metalloproteinase inhibitors (such as batimastatand marimastat), and those that are disclosed in WO2002055106; and (13)proteasome inhibitors, such as bortezomib (VELCADE).

The term “hormone therapeutic agent” refers to a chemical or biologicalsubstance that inhibits or eliminates the production of a hormone, orinhibits or counteracts the effect of a hormone on the growth and/orsurvival of cancer cells. Examples of such agents suitable for the VBIRinclude those disclosed in US20070117809. Examples of particular hormonetherapeutic agents include tamoxifen (NOLVADEX), toremifene (Fareston),fulvestrant (FASLODEX), anastrozole (ARIMIDEX), exemestane (AROMASIN),letrozole (FEMARA), megestrol acetate (MEGACE), goserelin (ZOLADEX),leuprolide (LUPRON), abiraterone, and MDV3100.

The VBIR provided by this disclosure may also be used in combinationwith other therapies, including (1) surgical methods that remove all orpart of the organs or glands which participate in the production of thehormone, such as the ovaries, the testicles, the adrenal gland, and thepituitary gland, and (2) radiation treatment, in which the organs orglands of the patient are subjected to radiation in an amount sufficientto inhibit or eliminate the production of the targeted hormone.

I. EXAMPLES

The following examples are provided to illustrate certain embodiments ofthe invention. They should not be construed to limit the scope of theinvention in any way. From the above discussion and these examples, oneskilled in the art can ascertain the essential characteristics of theinvention, and without departing from the spirit and scope thereof, canmake various changes and modifications of the invention to adapt it tovarious usage and conditions.

Example 1 Antigens in Cytosolic, Secreted, and Membrane-Bound FormatsDerived from the Human PSMA Protein

Example 1 illustrates the construction of three immunogenic PSMApolypeptides referred to an “human PSMA cytosolic antigen,” “human PSMAsecreted antigen,” and “human PSMA membrane-bound antigen,”respectively, and biological properties of these polypeptides.

1A. Design of Immunogenic PSMA Polypeptides

DNA constructs encoding immunogenic PSMA polypeptides in cytosolic,secreted, and modified formats were constructed based on the nativehuman PSMA protein sequence and tested for their ability to induceanti-tumor effector immune responses. The structure and preparation ofeach of the human PSMA antigen formats are provided as follows.

1A1. Human PSMA Cytosolic Antigen.

An immunogenic PSMA polypeptide in cytosolic form was designed to retainthe immunogenic polypeptide inside the cell once it is expressed. Thecytoplasmic domain (amino acids 1-19) and the transmembrane domain(amino acids 20-43) of the human PSMA were removed, resulting in acytosolic PSMA polypeptide that consists of amino acids 44-750(extracellular domain or ECD) of the human PSMA of SEQ ID NO: 1. Theoptimal Kozak sequence “MAS” may be added to the N-terminus of thepolypeptide for enhancing the expression.

1A2. Human PSMA secreted antigen. An immunogenic PSMA polypeptide insecreted form was designed to secret the polypeptide outside of the cellonce it is expressed. The secreted polypeptide is made with amino acids44-750 (ECD) of the human PSMA of SEQ ID NO:1 and the Ig Kappa secretoryelement that has the amino acid sequence ETDTLLLWVLLLWVPGSTGD and atwo-amino acid linker (AA) in the N-terminal in order to maximize thesecretion of the PSMA antigen once it is expressed.

1A3. Human PSMA membrane-bound antigen. An immunogenic PSMAmembrane-bound polypeptide was designed to stabilize the polypeptide onthe cell surface. The first 14 amino acids of the human PSMA proteinwere removed and the resultant immunogenic polypeptide consists of aminoadds 15-750 of the human PSMA protein of SEQ ID NO:1. The immunogenicpolypeptide that consists of amino adds 15-750 of the native human PSMAprotein of SES ID NO: 1 and share 100% sequence identity with the nativehuman PSMA protein is also referred to as “human PSMA modified,” “hPSMAmodified,” or “hPSMAmod” antigen in the present disclosure.

1B. Preparation of DNA Plasmids for Expressing the PSMA Antigens

DNA constructs encoding the PSMA cytosolic, PSMA secreted, and PSMAmodified antigens were cloned individually into PJV7563 vector that wassuitable for in vivo testing in animals (FIG. 1). Both strands of theDNA in the PJV7563 vectors were sequenced to confirm the designintegrity.

A large scale plasmid DNA preparation (Qiagen/CsCl) was produced from asequence confirmed clone. The quality of the plasmid DNA was confirmedby high 260/280 ratio, high super coiled/nicked DNA ratio, low endotoxinlevels (<10 U/mg DNA) and negative bio burden.

1C. Expression of PSMA Constructs in Mammalian Cells

The expression of the PSMA cytosolic, secreted, and modified antigenswas determined by FACS. Mammalian 293 cells were transfected with thePJV7563 PMED vectors encoding the various immunogenic PSMA polypeptides.Three days later, the 293 cells were stained with mouse anti-PSMAantibody, followed with a fluorescent conjugated (FITC) rat anti-mousesecondary antibody. The data below, which were reported as meanfluorescent intensity (MFI) over negative controls, confirmed that humanPSMA modified antigen is expressed on the cell surface.

Average mean Samples fluorescent intensity Untransfected 293 cells 231293 cells transfected with full length human 6425 PSMA (SEQ ID NO: 1)293 cells transfected with human PSMA 12270 modified antigen (SEQ ID NO:9)

1D. Formulations of PSMA Plasmids onto Gold Particles (for ND10/X15)

Particle Mediated Epidermal Delivery technology (PMED) is a needle-freemethod of administering vaccines to animals or to patients. The PMEDsystem involves the precipitation of DNA onto microscopic gold particlesthat are then propelled by helium gas into the epidermis. The ND10, asingle use device, uses pressurized helium from an internal cylinder todeliver gold particles and the ×15, a repeater delivery device, uses anexternal helium tank which is connected to the ×15 via high pressurehose to deliver the gold particles. Both of these devices were used instudies to deliver the PSMA DNA plasmids. The gold particle was usually1-3 μm in diameter and the particles were formulated to contain 2 μg ofPSMA plasmids per 1 mg of gold particles. (Sharpe, M. et al.: P.Protection of mice from H5N1 influenza challenge by prophylactic DNAvaccination using particle mediated epidermal delivery. Vaccine, 2007,25(34): 6392-98: Roberts L K, et al.: Clinical safety and efficacy of apowdered Hepatitis B nucleic acid vaccine delivered to the epidermis bya commercial prototype device. Vaccine, 2005; 23(40):4867-78).

1E. Transgenic Mice Used for In Vivo Studies

Two human HLA transgenic mouse models were used to evaluate thepresentation of various PSMA antigens by different HLAs and a human PSMAtransgenic mouse model was used to assess the breaking of immunetolerance to human PSMA. The first HLA transgenic mouse model utilizesthe HLA A2/DR1 mice (from the Pasteur Institute, Paris, France; alsoreferred to as “Pasteur mice”). Pasteur mice are knock out for murineβ-2-microglobulin and do not express functional H-2b molecules;therefore this model is believed to represent the presentation ofantigen in the human HLA A2 and DR1 context (Pajot, A., M.-L. Michel, N.Faxilleau, V. Pancre, C. Auriault, D. M. Ojcius, F. A. Lemonnier, andY.-C. Lone. A mouse model of human adaptive immune functions:HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice. Eur.J. Immunol. 2004, 34:3060-69.). The second HLA transgenic mouse modeluses mice that are knock in with human HLA A24 that is covalently linkedtothe human β-2-microglobulin at the H2bk locus. These mice lack murineβ-2-microglobulin and do not express functional H-2b molecules. Thismodel allows evaluation of antigen presentation in the context of humanHLA A24.

1F. Immunogenicity of the Human PSMA Proteins in Cytosolic, Secreted andModified Formats

Study Design.

Eight-to-10 week-old transgenic mice were immunized using PMED methodwith various PSMA DNA constructs in a prime/boost/boost regimen, twoweeks apart between each vaccination. Alternatively, mice were primedwith adenovirus vectors encoding the PSMA antigen at 1×10⁹ viralparticles in 50 μl (PBS) by intramuscular injection. The adenovirusvector (pShuttle-CMV vector from Stratagene) was modified to containNhel and Bglll restriction sites within the multiple cloning site. TheDNA encoding human PSMA modified was then restriction digested with Nheland BglII, ligated into this vector and sequence confirmed. The pShuttlehuman PSMA modified vector was then recombined with the pAdEasy-1 vectorand virus was propagated according to the AdEasy system (Stratagene).Twenty-days later, they were boosted with PMED as described above. Ineach of the regimens used, antigen specific T cell response was measured7 days after the last immunization in an interferon-gamma (IFNγ) ELISPOTassay. The ELISPOT assay is similar to the sandwich enzyme-linkedimmunosorbent assay (ELISA). Briefly, a capture antibody specific toIFNγ BD Bioscience, #51-2525kc) is coated onto a polyvinylidene fluoride(PVDF) membrane in a microplate overnight at 4° C. The plate is blockedwith serum/protein to prevent nonspecific binding to the antibody. Afterblocking, effector cells (such as splenocytes isolated from PSMAimmunized mice) and targets (such as PSMA peptides from peptide library,target cells pulsed with peptides or tumor cells expressing the relevantantigens) or mitogen (which will stimulate splenocytes non-specificallyto produce IFNγ are added to the wells and incubated overnight at 37° C.in a 5% CO₂ incubator. Cytokine secreted by effector cells are capturedby the coating antibody on the surface of the PVDF membrane. Afterremoving the cells and culture media, 100 μl of a biotinylatedpolyclonal anti-mouse IFNγ antibody (0.5 mg/ml-BD Bioscience,#51-1818kz) was added to each of the wells for detection. The spots arevisualized by adding streptavidin-horseradish peroxidase (HRP, BDBioscience, #557630) and the precipitate substrate,3-amino-9-ethylcarbazole (AEC), to yield a red color spot. Each spotrepresents a single cytokine producing T cell. In general, in thestudies disclosed here the ELISpot assay was set up as follows: 5×10⁵splenocytes from PSMA immunized mice were cultured (1) in the presenceof PSMA specific peptides derived from a PSMA peptide library (see Table16) made of 15-amino acid peptides overlapping by 11 amino acids, (2)with known HLA A2.1 restricted PSMA specific peptides, or (3) with tumorcells. To measure the recognition of endogenous antigen presentation,splenocytes were cultured with a human HLA A2 prostate cancer cells(i.e. LNCaP, available from ATCC) that naturally express PSMA orcultured with HLA A2 tumor cells transduced with adenovirus encoding andthus expressing the human PSMA modified antigen. In addition, human PSMAECD protein was added to the ELISpot assay to measure specifically CD4IFNγ producing cells. For controls where appropriate, HLA A2 restrictedHER-2 specific peptide p168-175 or tumor cells not expressing PSMA orirrelevant protein such as BSA were used as a negative control in theIFNγ ELISpot assay. Data results are given in normalized format for thenumber of spot forming cells (SFC) that secrete IFNγ in 1×10⁶splenocytes. At least three studies were performed for each of the PSMAantigen peptides tested.

Results.

Data from the ELISpot assay with splenocytes of Pasteur mice culturedwith peptides derived from a PSMA peptide library are presented inTable 1. A positive response is defined as having SFC >100. As shown inTable 1, the immunogenic PMSA polypeptides made with all three antigenformats, the human PSMA cytosolic, secreted, and modified antigensdescribed in Example 1A above, are capable of inducing T cell responses.The human PSMA modified antigen format induced the best breadth andmagnitude of T cell responses.

TABLE 1 T cell response induced by the human PSMA cytosolic, secreted,and modified antigens in Pasteur mice aa sequence coverage of targetpeptide pools from PSMA peptide IFN-γ SFC/1 × 10⁶ splenocytes (SD)library PSMA cytosolic PSMA modified PSMA secreted 13-35  1(14) 809(78)44(6)  97-117 1(1)  96(17) 0  109-131 8(9) 923(21) 179(24) 121-24321(4)  1329(109) 320(28) 145-167 23(4)  1499(1)  312(14) 169-191 497(4) 248(14) 183(13) 181-203 43(21)  70(20)  19(10) 205-227 5(1) 112(0)  9(13) 217-239 44(13) 1627(38)  351(10) 265-287 23(4)   527(100) 10(6)277-299 39(1)  1143(86)  151(4)  289-311 27(1)  429(4)   28(11) 409-43114(9)  281(18) 12(9) 421-443 4(0) 676(45)  48(14) 433-455 339(18) 713(64) 119(21) 481-503 22   288(9)   1(1) 577-599 227(27)  131(16) 33(16) 589-611 187(13)   27(10)  6(9) 613-635 418(6)  437(1)  55(1)637-659 222(31)   49(10)  95(16) 649-671 203(21)  1625(33)  420(11)661-683 102(14)  1633(140) 366(48) 697-719 179(4)  1357(58)  342(6) 709-731 40(11) 1162(59)  223(4)  721-743 56(6)  1409(103) 344(23)733-750 50(11) 1512(51)  365(27) ( ) = standard deviation

Data from the ELISpot assay on T cell responses induced by various PSMAvaccine formats in Pasteur mice (which that recognized HLA A2.1restricted PSMA peptide pulsed target cells as well as PSMA+HLA A2.1LNCaP tumor cells) are presented in Table 2. PC3, which is a humanprostate cancer cell line that does not express PSMA, was used here as anegative control. A positive response is defined as having SFC >50. Asshown in Table 2, the various PSMA constructs tested are capable ofinducing T cells that recognize known HLA A2 restricted PSMA epitopes aswell as PSMA protein and human prostate cancer cells LNCaP. However, thePSMA modified construct was shown to induce the best breadth andmagnitude T cell response.

TABLE 2 T cell responses induced by the human PSMA cytosolic, secreted,and modified antigens in Pasteur mice that recognized HLA A2.1restricted PSMA peptide pulsed target cells as well as PSMA+ HLA A2.1LNCaP tumor cells. HLA A2.1 restricted PSMA PSMA PSMA peptides cytosolicmodified secreted Target peptide or protein IFN-γ SFC/1 × 10⁶splenocytes PSMA p663 1554(4)   1524.9(45)   444(23) PSMA p275 14(10) 304(21)   3(1) PSMA p662 41(15) 925.1(77)  455(25) PSMA p627 1(1) 222(14)   9(8) PSA p64 0   2(4)  1(1) Protein or tumor cells IFN-γSFC/1 × 10⁶ splenocytes PSMA ECD protein 45(11) 731(16)  13(5)  LNCap4(2) 96(12) 5(3) PC3 0  1(1) 1(1) ( ) = standard deviation

1G. Humoral Immune Response Measured in Pasteur Mice or NonhumanPrimates

1G1. Sandwich ELISA Assay.

The standard sandwich ELISA assay was done using an automated Bioteksystem. The plates were coated with 25 μl of native PSMA protein at a1.0 μg/ml in PBS overnight, the plates were washed and blocked with 35μl/well of 5% FBS 1×PBS-T 0.05% and incubated for 1 hour at RT on ashaker at 600 RPM. The blocking media was decanted and serial dilutevaccinated mouse serum with half log dilutions in 5% FBS 1×PBS-T 0.05%starting at 1:100 or 1:500 were made and 25μ samples of the dilutedserum were added to each well of the 96 well plates and incubated for 1hour at RT on a shaker at 600 RPM. The plates were washed 3 times with75 ul/well in 1×PBS-T 0.05% using the Biotek ELx405, and 25 μl/well of1:30,000 diluted anti-mouse IgG HRP (AbCam cat# ab20043) secondaryantibody (diluted in 1× PBS-T 0.05%) was added to each well of the 96well plates and incubated for 1 hour at RT on a shaker at 600 RPM.Plates were washed 5× with 75 ul/well in 1×PBS-T 0.05% using the BiotekElx405. TMB Substrate was diluted at 1:10 and 25 μl was added to eachwell and incubated at RT for 30 minutes. The reaction was stopped byadding 12.5 μl/well of 1M H2504. Plates were read using the SpectramaxPlus at 450 nm wavelength. Data were reported as titers and these couldbe reported as first positive (average and both values above 5% FBSPBS+3 time Standard Deviation) and/or as calculated titers at OD of 0.5or 1.0. Serum from irrelevant vaccinated mice were used as negativecontrols.

TABLE 3 Induction of anti-PSMA antibody response by human PSMA antigensas measured by an ELISA assay. ELISA (OD = 1) Antigen format Average(+/−SD) N # of positive PSMA cytosolic 499 (0)  4 0/4 PSMA modified 1067(518) 4 4/4 PSMA secreted  959 (920) 4 1/4

Results.

Data presented in Table 3 shows that the human PSMA cytosolic antigendid not induce any anti-PSMA responses, while the human PSMA modifiedantigen consistently induced good anti-PSMA antibody responses in allmice.

Data presented in Table 5 shows that antibodies induced by the humanPSMA antigens reacted to multiple peptide epitopes in the PSMA library.Serum from the individual mice in each group was pooled in equal amountsand tested at a 1:500 dilution in an ELISA assay. A negative controlgroup of mice vaccinated with anti-diphtheria (CRM) toxoid was tested inparallel. Each well of the 96 well ELISA plate was coated with 0.03 μgof a single15aa peptide derived from the PSMA peptide library. An ODvalue above 0.10 is considered positive.

1G2. FACS Cell Binding Assay.

Various prostate cancer cell lines were used for this assay. LNCaP(ATCC) was used as human prostate cancer cells expressing PSMA and PC3(ATCC) was used as negative human prostate cancer cells that do notexpressing PSMA. In some assays, a TRAMP-C2 cell line engineered tostably express the human native full length PSMA and the parentalTRAMP-C2 cell line that does not express PSMA (negative control) wereused for the cell binding assay. The cell binding assay was performed asfollows: LNCaP and PC3 cells (or TRAMP-C2PSMA and TRAMP C2) were platedin separate wells at 2×10⁵ cells/well (50 μL) in a 96 well plate. Serafrom PSMA vaccinated mice, as described in 1f, were diluted 1:50 withFACS buffer (PBS pH 7.4, 1% FBS, 25 mM HEPES, and 1 mM EDTA). Fifty μLof diluted J591-A antibody (mouse anti-human PSMA antibody, clone J591-Afrom ATCC) were added to the diluted test sera or FACS buffer (unstainedsamples) to achieve the appropriate cell numbers per well in thestaining plate. All was mixed by pipetting and then kept on ice for 20min. The cells were washed twice with FACS buffer; each wash was bycentrifugation at 1200RPM at 5° C. for 5 minutes. Fifty μL of secondarystaining solution were added containing a 1:200 dilution of PE-labeledgoat anti-mouse Ig (Sigma, cat P9670-5) and 0.25 μl of Live/Dead Aquastain (Invitrogen, cat. # L34957) to each of the cell containing wellsand kept on ice for 20 min. Cells were washed twice as describedearlier. Washed cell pellets were resuspended in 125 uL FACS buffer andthen 75 uL 4% paraformaldehyde solution were added to each well to fixthe cells. Samples were kept on ice and protected from light for atleast 15 min. Samples were run on FACS Canto II. Ten thousand live cellevents were recorded for each sample. Control samples for each cell typewere 1) unstained cells, 2) cells with secondary antibody only, 3) Cellswith J591 plus secondary antibody, and 4) cells with naïve serum plussecondary antibody. Data were reported as mean fluorescent intensity(MFI) over negative controls.

Results of FACS Cell Binding Assay.

Table 4 shows that antibodies induced by both human PSMA secreted andmodified antigens are capable of binding to human PSMA positive prostatecancer cells (LNCaP) and not to PSMA negative prostate cancer cells(PC3). The PSMA modified antigen consistently induced good anti-PSMAantibody response in all mice.

TABLE 4 Binding of anti-PSMA antibodies to human prostate cancer cellsas measured by FACS. Fold over background Antigen Format Average (+/−SD)N # of positive PSMA cytosolic 1.40 (0.12) 4 0/4 PSMA modified 6.01(0.38) 4 4/4 PSMA secreted 5.50 (4.10) 4 3/4 background (PC3) 1.36(0.04) 4 NA

TABLE 5 Antibodies induced by PSMA vaccine reacted to multiple peptidesin the PSMA library. Based on this result, four B cell epitopes of PSMAwere identified, 1: aa 138-147, 2: aa 119-123, 3: aa 103-106, 4: aa649-659. ELISA O.D. results PSMA vaccinated CRM-197 Target peptide orprotein serum vaccinated serum Peptides  93 0.21 0.05 from PSMA 101 0.270.05 library (no. 133 0.58 0.05 of first aa) 137 0.89 0.05 141 0.12 0.05645 0.17 0.05 PSMA protein 3.87 0.05

1 G3. Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Assay

Study Design.

An Indian rhesus macaque was immunized with a nucleic acid encoding ahuman PSMA modified antigen delivered by adenovirus (1e11 V.P. injectedintramuscularly) followed by 2 PMED immunizations (8actuations/immunization, 4 actuations per each right and left side ofthe lower abdomen) with 8 and 6 week intervals respectively. The animalalso received intradermal injections of 3 mg of CpG (PF-03512676) inproximity to each inguinal draining lymph node at the time of the secondPMED immunization. The antibody dependent cell-mediated cytotoxicity wasdetermined from the plasma collected from the blood before anyimmunizations (pre-immune plasma) and 8 days after the last PMEDimmunization (immune plasma).

Antibody-Dependent Cell-Mediated Cytotoxicity Assay.

Antibody-dependent cell-mediated cytotoxicity was determined using thestandard chromium 51 release assay. Human prostate cancer cell linesLNCaP and PC3 were used as target cells. Freshly isolated human PBMCcells were used as effector cells. Effectors to target cells were set at30:1. Briefly, for one labeling reaction, 1.5×10⁶ target cells in 200 ulwere incubated with 200 μCi ⁵¹Cr (37° C., 5% CO₂ for 1 hour). Cells werewashed three times and the cell concentration was adjusted to 2×10⁵cells/ml. Control monoclonal antibodies (mAb) or test plasma (1:50) weremade at 2× concentration and 175 ul of each of the (depending on thesize of the assay) mAb/plasma dilution were added to 175 ul of targetcells. The mixture was incubated for 30 minutes at 4° C. in an Eppendorftube. Cells were washed once to free unbound antibodies. At this time,100 ul of freshly isolated effector cells were added to each well of the96 well plate along with 100 ul of monoclonal antibodies or test plasmabound target cells and incubated at 37° C. and 5% CO₂ for 4 hrs. Sampleswere tested in duplicates. 100 μl 2N HCl were added to the target wellsfor maximum release and 100 μl of media were added to the target wellsfor spontaneous release. Specific lysis was calculated as follows:Percent release=(ER−SR)/(MR−SR)×100 where ER (effectors+target cellsrelease) was experimental release, SR (target cells alone incubated withmedia) was spontaneous release, and MR (target cells alone incubatedwith 2N HCl) was maximum release. Percent specific lysis was calculatedby subtracting irrelevant target (PC3) release from antigen specifictarget (LNCaP) release.

TABLE 6 Antibody dependent cytotoxicity activity measured in plasma fromhuman PSMA modified vaccinated animal. Percent specific lysis at E:T of30:1 (based on type of antibodies and targets used in an ADCC assay)Pre-immune Post-immune Targets Herceptin Rituxan plasma (naive) plasmaNone LNCaP 56.14 8.99 0 49.9 0 PC3 8.04 0 0 0 0

Results.

The data from the antibody dependent cytotoxicity assay are presented inTable 6. LNCaP, a human prostate PSMA+ cancer cell line coated withimmune plasma derived from the hPSMA immunized animal, was lysed byeffector cells while PC3, a human prostate PSMA-cancer cell line coatedwith the same immune serum, was not lysed by effector cells. Similarly,LNCaP coated with pre-immune plasma was not lysed by effector cells.Herceptin, a monoclonal antibody against HER-2 was used as a positivecontrol since LNCaP cells are known to express HER-2 (Li, Cozzi et al.2004). Rituxan, a monoclonal against B cell antigen (CD20) was used as anegative control antibody since LNCaP cells do not express CD20. Bothmonoclonal antibodies are reported to have ADCC activities (Dall′Ozzo,Tartas et al. 2004; Collins, O'Donovan et al. 2011).

Example 2 Construction of PSMA Shuffled Antigens

This example illustrates the construction and certain biologicalproperties of various immunogenic PSMA polypeptides that are variants ofthe human PSMA modified antigen (SEQ ID NO:9) as described in Example 1.

2A. Design of PSMA Shuffled Antigens

Various immunogenic PSMA polypeptides that are variants of the humanPSMA modified antigen (SEQ ID NO:9) as described in Example 1 weredesigned. These variants were created by introducing mutations selectedfrom orthologs of the human PSMA into the human PSMA modified antigensequence. These variants are referred to, interchangeably, as “PSMAshuffled antigens” or “shuffled PSMA modified antigens” in thedisclosure. The principle and procedure used in creating these variantsare provided below.

A computational algorithm was written to select point mutations for theshuffled variant. First, a multiple sequence alignment of PSMA and 12orthologs (Appendix 2a) was assembled using NCBI's PSI-BLAST. The outputfrom PSI-BLAST included propensities for each residue at each PSMAposition among the orthologs. The perl script then used thesepropensities to select point mutations as follows:

1) Among all positions, the most commonly observed residue is selectedthat does not match the identity in the native human PSMA.

2) Verify that this mutation position does not overlap with identifiedClass I or II human PSMA epitopes to ensure that the point mutation isnot within a conserved T cell epitope as defined herein above (Table19).

3) Calculate similarity of mutation to the human residue via theBLOSUM62 matrix to verify that the BLOSUM62 similarity score for theresidue substitution is within the range of 0-1 (inclusive).

This iterative procedure is followed until a certain percent sequenceidentity (below 100) is reached with respect to the human PSMA.

To serve as the input to this algorithm, the PSMA orthologs wereassembled to construct a position-specific probability matrix usingPSI-BLAST from NCBI. Additionally, the identified epitope regions ofPSMA were listed in a file which was also provided to the shufflealgorithm. The non-shuffling regions were also extended to the cytosolicand transmembrane regions of the protein to avoid membrane-boundfunctionality problems. The orthologous PSMA protein sequences, BLOSUM62matrix, and PSI-BLAST program were downloaded from the NCBI site.

The shuffling script was then run using these input data and produced avariant of human PSMA with 94% sequence identity with the original humanPSMA. Additionally, three mutations to improve HLA-A2 binding wereintroduced based on their performance in the Epitoptimizer algorithm(Houghton, Engelhorn et al. 2007). These mutations are M664L (epitope:663-671), 1676V (epitope: 668-676), and N76L (epitope: 75-83). Theresultant antigen is referred to as “shuffled PSMA modified antigen 1,”“shuffled PSMA modified 1,” or “PSMA shuffled antigen 1”.

Results based on epitopes with consensus rank <1% and IC50 by neuralNetwork (single best method)<500 showed that predicted epitopes from HLAA2.1, HLA A3, HLA A11, HLA A24, and HLA B7 were highly conserved in thisshuffled antigen. Two additional variants of the human PSMA modifiedantigen described in Example 1 were designed ‘with higher sequenceidentities and a more restrictive BLOSUM score cutoff of 1 to remove allnon-conservative substitutions. These two variants are also referred toas “shuffled PSMA modified antigen 2” and “shuffled PSMA modifiedantigen 3,” respectively. Percent identities of shuffled PSMA modifiedantigens 1-3 with respect to the human PSMA modified construct (e.g.,amino acids 15-750 of the human PSMA) are approximately 93.6%, 94.9%,and 96.4%, respectively.

The shuffled PSMA modified antigen 1 has the amino acid sequence of SEQID NO:3 and has the following mutations relative to the human PSMAmodified antigen: N47S, T53S, K55Q, M58V, L65M, N76L, S98A, Q99E, K122E,N132D, V154I, I157V, F161Y, D191E, M192L, V201L, V225I, I258V, G282E,I283L, R320K, L3621, S380A, E408K, L4171, H475Y, K482Q, M509V, S513N,E542K, M583L, N589D, R598Q, S613N, I614L, S615A, Q620E, M622L, S647N,E648Q, S656N, I659L, V660L, L661V, M664L, I676V

The shuffled PSMA modified antigen 2 has the amino acid sequence of SEQID NO:5 and has the following mutations relative to the human PSMAmodified antigen: Mutations: N47S, K55Q, M58V, Q91E, S98A, A111S, K122E,N132D, V154I, I157V, F161Y, V201L, V225I, I258V, S312A, R320K, K324Q,R363K, S380A, E408K, H475Y, K482Q, Y494F, E495D, K499E, M509L, N540D,E542K, N544S, M583I, I591V, R598Q, R605K, S613N, S647N, E648Q, S656N,V660L

The shuffled PSMA modified antigen 3 has the amino acid sequence of SEQID NO:7 and has the following mutations relative to the human PSMAmodified antigen: Mutations: T339A, V342L, M344L, T349N, N350T, E351K,S401 T, E408K, M470L, Y471H, H475Y, F506L, M509L, A531S, N540D, E542K,N544S, G548S, V555I, E563V, V603A, R605K, K606N, Y607H, D609E, K610N,I611L

2B. Immune Responses Measured Post Vaccination in Pasteur Mice

Study Design.

Eight- to 10-week old Pasteur mice were immunized using PMED method withthe various plasmid DNAs expressing shuffled PSMA modified antigens in aprime/boost/boost regimen, two weeks apart between each vaccination asdescribed in Example 1F. Antigen specific T and B cell responses weremeasured 7 days after the last immunization in an interferon-gamma(IFNγ) ELISPOT assay and sandwich ELISA respectively.

TABLE 7 T cell responses induced by various shuffled PSMA modifiedantigens to peptide pools in PSMA peptide library Amino acid IFN-γ SFC/1× 10⁶ splenocytes (SD) sequence coverage Human Shuffled ShuffledShuffled of target peptide PSMA PSMA PSMA PSMA pools from PSMA modifiedmodified modified modified peptide library antigen antigen1 antigen 2antigen 3 13-35 608 (31) 135 (4) 539 (33) 339 (16) 109-131 132 (14) 97(21) 49 (13) 360 (40) 121-243 275 (7) 146 (17) 104 (17) 542 (119)145-167 218 (6) 158 (8) 98 (8) 505 (16) 157-179 212 (14) 293 (24) 800(0)* 800 (0)* 169-191 50 (11) 52 (14) 814 (23) 804 (65) 181-203 415 (33)8 (0) 243 (18) 103 (1) 205-227 125 (7) 1 (1) 17 (7) 7 (4) 217-239 883(47) 302 (0) 467 (52) 538 (14) 229-251 565 (24) 188 (23) 150 (17) 460(122) 265-287 418 (8) 2 (0) 154 (25) 168 (23) 277-299 908 (79) 132 (3)574 (25) 670 (74) 289-311 417 (27) 20 (8) 260 (3) 374 (51) 409-431 377(7) 61 (10) 38 (3) 48 (11) 421-443 720 (34) 110 (9) 720 (17) 38 (17)433-455 974 (51) 211 (16) 800 (0)* 771 (52) 481-503 400 (59) 0 (0) 116(6) 100 (34) 589-611 60 (14) 245 (30) 679 (35) 364 (11) 601-623 70 (9)79 (13) 344 (20) 27 (1) 613-635 629 (41) 102 (11) 772 (93) 634 (9)637-659 226 (17) 292 (0) 539 (16) 420 (198) 649-671 530 (74) 319 (16)614 (20) 644 (65) 661-683 507 (52) 248 (9) 330 (3) 661 (16)

Results.

ELISpot data presented in Table 7 demonstrates that overall the shuffledPSMA modified antigens are capable of inducing T cell responses inbreadth and magnitude very similar to the human PSMA modified antigen.SFC >100 is considered positive. The “*” symbol represents too numerousto accurately count.

TABLE 8 T cell responses induced by shuffled PSMA modified antigens toHLA A2 targets and PSMA protein. IFN-γ SFC/1 × 10⁶ splenocytes(SD) HumanShuffled Shuffled Shuffled PSMA PSMA PSMA PSMA IFNγ ELISPOT modifiedmodified modified modified targetstargets antigen antigen 1 antigen 2antigen 3 PSMA p168 261 (21) 337 (16) 800+ (0) 800+ (0) PSMA p275 540(66) 2 (2) 181 (17) 134 (17) PSMA p663 441 (46) 152 (10) 219 (6) 600(48) HER-2 p106 1 (1) 1 (1) 1 (1) 1 (1) PSMA ECD protein 839 (70) 165(31) 569 (44) 319 (6) BSA protein 1 (1) 1 (1) 1 (1) 2 (0) LNCaP 229 (45)40 (3) 137 (23) 45 (6) MeWo 3 (1) 1 (1) 2 (2) 1 (1) MeWo-Ad-hPSMA 365(33) 31 (10) 341 (46) 415 (128)

As shown in Table 8, all the shuffled PSMA antigens are capable ofinducing T cells that recognized known HLA A2 restricted PSMA epitopesas well as human HLA A2 tumor cells transduced with adenovirus bearingthe PSMA transgene to express PSMA. The tumor cells that did not expressPSMA served as negative controls and were not recognized. SFC>50 isconsidered positive.

TABLE 9 T cell responses induced by shuffled PSMA modified antigens tospecific to CD4 T cells. aa sequence IFN-γ SFC/1 × 10⁶ CD8 depletedsplenocytes(SD) coverage of Human Shuffled Shuffled Shuffled targetpeptide PSMA PSMA PSMA PSMA pools from PSMA modified modified modifiedmodified peptide library antigen antigen 1 antigen 2 antigen 3 17-31 396(51) 49 (33) 294 (3) 209 (13) 281-295 528 (6) 51 (1) 592 (9) 461 (24)285-299 512 (34) 55 (18) 529 (38) 471 (38) 429-443 552 (51) 51 (1) 524(17) 0 581-595 11 (1) 0 199 (24) 0

ELISpot data shown in Table 9 were obtained with splenocytes that weredepleted of CD8; therefore the data represents T cell responses tospecific to CD4 T cells. The data show that the CD4 response elicited byshuffled PSMA modified antigen 2 is very similar to that induced by thehuman PSMA modified antigen. SFC>50 is considered positive.

TABLE 10 Induction of anti-PSMA antibody response as measured by anELISA assay Antigen ELISA (OD = 0.5) # of positive Human PSMA modifiedantigen 1159 (802)  7/7 Shuffled PSMA modified antigen 1  899 (1016) 4/7Shuffled PSMA modified antigen 2 4898 (3636) 6/6 Shuffled PSMA modifiedantigen 3 1482 (3092) 1/5

Data in Table 10 demonstrates that all the shuffled PSMA modifiedantigens are capable of inducing anti-human PSMA antibody responses.Shuffled PSMA modified antigen 2 and the human PSMA modified antigeninduced consistent antibody responses in all mice.

TABLE 11 T cell responses in HLA A24 mice induced by the human PSMAmodified antigen and shuffled PSMA modified antigen 2. Amino acidsequence IFN-γ SFC/1 × 10⁶ splenocytes (SD) coverage of target HumanShuffled peptide pools from PSMA modified PSMA modified PSMA peptidelibrary antigen antigen 2 217-231 280 (10) 271 (7) 233-247 219 (2) 0237-251 197 (1) 228 (1) 249-263 203 (1) 28 (1) 273-287 57 (2) 323 (3)277-291 194 (24) 337 (18) 293-307 147 (6) 379 (11) 309-323 17 (1) 441(14) 401-415 256 (1) 292 (3) 429-443 255 (7) 0 433-447 59 (1) 179 (6)481-495 167 (11) 475 (21) 557-571 194 (14) 297 (1) 601-615 500 (35) 166(10) 605-619 500 (28) 143 (7) 613-627 218 (4) 0 697-711 0 141 (8)729-743 0 140 (1) 733-747 0 271 (3) 737-750 0 401 (8)

ELISpot data in Table 11 demonstrates that overall the T cell responseinduced by shuffled PSMA modified antigen 2 in HLA A24 mice is verysimilar in breadth and magnitude to the human PSMA modified antigen.SFC >100 is considered positive.

2C. Breaking of Immune Tolerance to Human PSMA by Shuffled PSMA ModifiedAntigens

Study Design.

The human PSMA transgenic mouse model uses mice that were made using theminimal rat probasin promoter driving the expression of PSMAspecifically in the prostate gland (Zhang, Thomas et al. 2000)Endocrinology 141(12): 4698-4710. These mice were made in the C57BL/6background. RT-PCR and immune histochemistry staining data confirmed theexpression of PSMA in the ventral and dorsolateral roots of the prostategland in these PSMA transgenic mice. The endogenous expression of humanPSMA protein in these mice is expected to generate immune tolerance.

Results.

As shown in Table 12, only 20% of the PSMA transgenic mice were able tomount a T cell response to human PSMA using the human PSMA modifiedantigen. However, 67% of the PSMA transgenic mice were able to mount aPSMA specific T cell response using the shuffled PSMA modified antigen2. The data suggests that the inclusion of non-self amino acid sequencesin the shuffled PSMA modified antigen 2 improved the breaking toleranceto the self human PSMA antigen. SFC>50 is considered positive.

TABLE 12 T cell responses in human PSMA transgenic mice to known PSMAepitope (PADYFAPGVKSYPDG; Durso R J, Clin Cancer Res. 2007 Jul. 1;13(13): 3999-400). # of Antigen IFN-γ SFC/1 × 10⁶ splenocytes (SD)positive Human 7 126 1 3 10 22 30 12 7 404 2/10 PSMA (3) (20) (2) (1)(5) (5) (3) (0) (2) (4) modified antigen Shuffled 474 104 7 111 12 21139 246 226 NA 6/9  PSMA (52) (18) (3) (8) (2) (2) (12) (9) (11)modified antigen 2

Example 3 Design of Various Immunogenic PSA Polypeptides

Example 3 illustrates the construction and certain biological propertiesof immunogenic PSA polypeptides in cytosolic, secreted, andmembrane-bound forms.

3A. Construction of Various PSA Antigen Forms

Similar to what was described in Example 1 for the three differentimmunogenic PSMA polypeptide forms (e.g., the cytosolic, membrane-bound,and secreted forms), immunogenic PSA polypeptides in the three formswere also designed based on the human PSA sequence. An immunogenic PSApolypeptide in cytosolic form, which consists of amino acids 25-261 ofthe native human PSA, is constructed by deleting the secretory signaland the pro domain (amino acids 1-24). The amino acid sequence of thiscytosolic immunogenic PSA polypeptide is provided in SEQ ID NO: 17. Thesecreted form of the PSA polypeptide is the native full length human PSA(amino acids 1-261). An immunogenic PSA polypeptide in membrane-boundform is constructed by linking the immunogenic PSA polypeptide cytosolicform (amino acids 25-261 of the native human PSA) to the human PSMAtransmembrane domain (amino acids 15-54 of the human PSMA).

3B. Immune Responses in Pasteur and HLA A24 Mice

Study Design.

Eight to 10 week old HLA A2 Pasteur mice or HLA A24 mice were immunizedwith DNA expressing the various PSA antigens using PMED provided inExample 3A in a prime/boost/boost regimen with two week intervalsbetween each vaccination as described in Example 1. The antigen specificT and B cell responses were measured 7 days after the last immunizationin an interferon-gamma (IFNγ) ELISPOT assay and sandwich ELISA.

TABLE 13 Induction of T cell responses in Pasteur mice and HLA A24 micevaccinated with PSA polypeptides Amino acid sequence coverage of targetIFN-γ SFC/1 × 10⁶ splenocytes (SD) peptide pools from PSA PSA PSA PSApeptide library cytosolic membrane-bound secreted T cell responsedetected in HLA A2 Pasteur mice 25-47 12 (0) 134 (6) 27 (1) 49-71 150(0) 23 (1) 151 (10) 61-83 904 (17) 27 (7) 452 (14) 73-95 128 (25) 8 (6)78 (14)  85-107 17 (7) 205 (18) 11 (1)  97-117 26 (3) 378 (25) 13 (1)217-239 16 (8) 234 (8) 6 (6) 229-251 96 (34) 844 (6) 35 (12) T cellresponse detected in HLA A24 mice 145-167 357 (2) Not Not determineddetermined

Results.

Table 13 shows ELISpot data derived from splenocytes isolated from HLAA2 Pasteur mice or HLA A24 mice cultured with peptides derived from thePSA peptide library. T cell responses can be detected in both HLA A2 andHLA A24 mice. SFC>100 is considered positive.

TABLE 14 The induction of T cell responses by PSA antigens in Pasteurmice to PSA+ HLA A2.1+ SKmel5 human cancer cells IFN-γ SFC/1 × 10⁶splenocytes (SD) HLA A2.1+ human PSA membrane- cancer cells or proteinPSA cytosolic bound PSA secreted SKmel5-Ad-eGFP 7.7 (9.6) 1.2 (1.4) 2.9(2.7) SKmel5-Ad-PSA 112.0 (169.3) 546.1 (379.6) 18.7 (18.5) PSA protein108.8 (161.0) 536.9 (380.9) 20.6 (21)

ELISpot data shown in table 14 indicates that immunogenic PSApolypeptides in both cytosolic and membrane-bound forms are capable ofinducing T cells that recognize human tumor cells transduced withadenovirus to express the cytosolic PSA antigen (SKmel5-Ad-PSA) but notcells transduced with adenovirus to express eGFP (SKmel5-Ad-eGFP). Thesetwo antigens also elicited response to PSA protein. The PSA secretedantigen failed to induce T cells to both SKmel5-Ad-PSA or PSA protein.SFC>50 is considered positive.

TABLE 15 The induction of anti-PSA antibody response as measured by asandwich ELISA assay ELISA (OD = 1.0) Antigen Forms Average (SD) # ofpositive PSA cytosolic 99 (0) 0/6 PSA membrane-bound 4838 (835) 6/6 PSAsecreted 1151 2410) 2/6

Data in Table 15 demonstrates that immunogenic PSA polypeptides in bothsecreted and membrane-bound forms are capable of inducing anti-PSAantibody responses.

TABLE 16 Human PSMA Peptide Library peptide pools and correspondingamino acid sequences PSMA peptide aa sequences of individual pool (aano.) peptides  1-23 MWNLLHETDSAVATA LHETDSAVATARRPR DSAVATARRPRWLCA13-35 ATARRPRWLCAGALV RPRWLCAGALVLAGG LCAGALVLAGGFFLL 25-47ALVLAGGFFLLGFLF AGGFFLLGFLFGWFI FLLGFLFGWFIKSSN 37-59 FLFGWFIKSSNEATNWFIKSSNEATNITPK SSNEATNITPKHNMK 49-71 ATNITPKHNMKAFLD TPKHNMKAFLDELKANMKAFLDELKAENIK 61-83 FLDELKAENIKKFLY LKAENIKKFLYNFTQ NIKKFLYNFTQIPHL73-95 FLYNFTQIPHLAGTE FTQIPHLAGTEQNFQ PHLAGTEQNFQLAKQ  85-107GTEQNFQLAKQIQSQ NFQLAKQIQSQWKEF AKQIQSQWKEFGLDS  97-117 QSQWKEFGLDSVELAKEFGLDSVELAHYDV LDSVELAHYDVLLSY 109-131 ELAHYDVLLSYPNKT YDVLLSYPNKTHPNYLSYPNKTHPNYISII 121-243 NKTHPNYISIINEDG PNYISIINEDGNEIF SIINEDGNEIFNTSL133-155 EDGNEIFNTSLFEPP EIFNTSLFEPPPPGY TSLFEPPPPGYENVS 145-167EPPPPGYENVSDIVP PGYENVSDIVPPFSA NVSDIVPPFSAFSPQ 157-179 IVPPFSAFSPQGMPEFSAFSPQGMPEGDLV SPQGMPEGDLVYVNY 169-191 MPEGDLVYVNYARTE DLVYVNYARTEDFFKVNYARTEDFFKLERD 181-203 RTEDFFKLERDMKIN FFKLERDMKINCSGK ERDMKINCSGKIVIA193-215 KINCSGKIVIARYGK SGKIVIARYGKVFRG VIARYGKVFRGNKVK 205-227YGKVFRGNKVKNAQL FRGNKVKNAQLAGAK KVKNAQLAGAKGVIL 217-239 AQLAGAKGVILYSDPGAKGVILYSDPADYF VILYSDPADYFAPGV 229-251 SDPADYFAPGVKSYP DYFAPGVKSYPDGWNPGVKSYPDGWNLPGG 241-263 SYPDGWNLPGGGVQR GWNLPGGGVQRGNIL PGGGVQRGNILNLNG253-275 VQRGNILNLNGAGDP NILNLNGAGDPLTPG LNGAGDPLTPGYPAN 265-287GDPLTPGYPANEYAY TPGYPANEYAYRRGI PANEYAYRRGIAEAV 277-299 YAYRRGIAEAVGLPSRGIAEAVGLPSIPVH EAVGLPSIPVHPIGY 289-311 LPSIPVHPIGYYDAQ PVHPIGYYDAQKLLEIGYYDAQKLLEKMGG 301-323 DAQKLLEKMGGSAPP LLEKMGGSAPPDSSW MGGSAPPDSSWRGSL313-335 APPDSSWRGSLKVPY SSWRGSLKVPYNVGP GSLKVPYNVGPGFTG 325-347VPYNVGPGFTGNFST VGPGFTGNFSTQKVK FTGNFSTQKVKMHIH 337-359 FSTQKVKMHIHSTNEKVKMHIHSTNEVTRI HIHSTNEVTRIYNVI 349-371 TNEVTRIYNVIGTLR TRIYNVIGTLRGAVENVIGTLRGAVEPDRY 361-383 TLRGAVEPDRYVILG AVEPDRYVILGGHRD DRYVILGGHRDSWVF373-395 ILGGHRDSWVFGGID HRDSWVFGGIDPQSG WVFGGIDPQSGAAVV 385-407GIDPQSGAAVVHEIV QSGAAVVHEIVRSFG AVVHEIVRSFGTLKK 397-419 EIVRSFGTLKKEGWRSFGTLKKEGWRPRRT LKKEGWRPRRTILFA 409-431 GWRPRRTILFASWDA RRTILFASWDAEEFGLFASWDAEEFGLLGS 421-443 WDAEEFGLLGSTEWA EFGLLGSTEWAEENS LGSTEWAEENSRLLQ433-455 EWAEENSRLLQERGV ENSRLLQERGVAYIN LLQERGVAYINADSS 445-467RGVAYINADSSIEGN YINADSSIEGNYTLR DSSIEGNYTLRVDCT 457-479 EGNYTLRVDCTPLMYTLRVDCTPLMYSLVH DCTPLMYSLVHNLTK 469-491 LMYSLVHNLTKELKS LVHNLTKELKSPDEGLTKELKSPDEGFEGK 481-503 LKSPDEGFEGKSLYE DEGFEGKSLYESWTK EGKSLYESWTKKSPS493-515 LYESWTKKSPSPEFS WTKKSPSPEFSGMPR SPSPEFSGMPRISKL 505-527EFSGMPRISKLGSGN MPRISKLGSGNDFEV SKLGSGNDFEVFFQR 517-539 SGNDFEVFFQRLGIAFEVFFQRLGIASGRA FQRLGIASGRARYTK 529-551 GIASGRARYTKNWET GRARYTKNWETNKFSYTKNWETNKFSGYPL 541-563 WETNKFSGYPLYHSV KFSGYPLYHSVYETY YPLYHSVYETYELVE553-575 HSVYETYELVEKFYD ETYELVEKFYDPMFK LVEKFYDPMFKYHLT 565-587FYDPMFKYHLTVAQV MFKYHLTVAQVRGGM HLTVAQVRGGMVFEL 577-599 AQVRGGMVFELANSIGGMVFELANSIVLPF FELANSIVLPFDCRD 589-611 NSIVLPFDCRDYAVV LPFDCRDYAVVLRKYCRDYAVVLRKYADKI 601-623 AVVLRKYADKIYSIS RKYADKIYSISMKHP DKIYSISMKHPQEMK613-635 SISMKHPQEMKTYSV KHPQEMKTYSVSFDS EMKTYSVSFDSLFSA 625-647YSVSFDSLFSAVKNF FDSLFSAVKNFTEIA FSAVKNFTEIASKFS 637-659 KNFTEIASKFSERLQEIASKFSERLQDFDK KFSERLQDFDKSNPI 649-671 RLQDFDKSNPIVLRM FDKSNPIVLRMMNDQNPIVLRMMNDQLMFL 661-683 LRMMNDQLMFLERAF NDQLMFLERAFIDPL MFLERAFIDPLGLPD673-695 RAFIDPLGLPDRPFY DPLGLPDRPFYRHVI LPDRPFYRHVIYAPS 685-707PFYRHVIYAPSSHNK HVIYAPSSHNKYAGE APSSHNKYAGESFPG 697-719 HNKYAGESFPGIYDAAGESFPGIYDALFDI FPGIYDALFDIESKV 709-731 YDALFDIESKVDPSK FDIESKVDPSKAWGESKVDPSKAWGEVKRQ 721-743 PSKAWGEVKRQIYVA WGEVKRQIYVAAFTV KRQIYVAAFTVQAAA733-750 YVAAFTVQAAAETLS FTVQAAAETLSEVA

TABLE 17 Human PSA Peptide Library PSA peptide aa sequences ofindividual pool (aa no.) peptides  1-23 MWVPVVFLTLSVTWI VVFLTLSVTWIGAAPTLSVTWIGAAPLILS 13-35 TWIGAAPLILSRIVG AAPLILSRIVGGWEC ILSRIVGGWECEKHS25-47 IVGGWECEKHSQPWQ WECEKHSQPWQVLVA KHSQPWQVLVASRGR 37-59PWQVLVASRGRAVCG LVASRGRAVCGGVLV RGRAVCGGVLVHPQW 49-71 VCGGVLVHPQWVLTAVLVHPQWVLTAAHCI PQWVLTAAHCIRNKS 61-83 LTAAHCIRNKSVILL HCIRNKSVILLGRHSNKSVILLGRHSLFHP 73-95 ILLGRHSLFHPEDTG RHSLFHPEDTGQVFQ FHPEDTGQVFQVSHS 85-107 DTGQVFQVSHSFPHP VFQVSHSFPHPLYDM SHSFPHPLYDMSLLK  97-117PHPLYDMSLLKNRFL YDMSLLKNRFLRPGD LLKNRFLRPGDDSSH 109-131 RFLRPGDDSSHDLMLPGDDSSHDLMLLRLS SSHDLMLLRLSEPAE 121-243 LMLLRLSEPAELTDA RLSEPAELTDAVKVMPAELTDAVKVMDLPT 133-155 TDAVKVMDLPTQEPA KVMDLPTQEPALGTT LPTQEPALGTTCYAS145-167 EPALGTTCYASGWGS GTTCYASGWGSIEPE YASGWGSIEPEEFLT 157-179WGSIEPEEFLTPKKL EPEEFLTPKKLQCVD FLTPKKLQCVDLHVI 169-191 KKLQCVDLHVISNDVCVDLHVISNDVCAQV HVISNDVCAQVHPQK 181-203 NDVCAQVHPQKVTKF AQVHPQKVTKFMLCAPQKVTKFMLCAGRWT 193-215 TKFMLCAGRWTGGKS LCAGRWTGGKSTCSG RWTGGKSTCSGDSGG205-227 GKSTCSGDSGGPLVC CSGDSGGPLVCNGVL SGGPLVCNGVLQGIT 217-239LVCNGVLQGITSWGS GVLQGITSWGSEPCA GITSWGSEPCALPER 229-251 WGSEPCALPERPSLYPCALPERPSLYTKVV PERPSLYTKVVHYRK 241-263 SLYTKVVHYRKWIKD KVVHYRKWIKDTIVAYRKWIKDTIVANP

TABLE 18 PSMA Orthologs PSMA species NCBI ID % ID with human human2897946 100 chimpanzee 114639743 99 macaque 109108238 97 dog 73987958 93horse 149719573 92 pig 47523822 90 cow 156120365 89 rat 149069047 84mouse 20138153 84 opossum 126327828 80 chicken 118085215 78 platypus149635150 76 zebra fish 41053648 69

TABLE 19 Conserved T Cell Epitopes in the Human PSMA as Set Forth in SEQID NO: 1. Amino acid Start Amino acid End Sequence 168 176 GMPEGDLVY 347356 HSTNGVTRIY 557 566 ETYELVEKFY 207 215 KVFRGNKVK 431 440 STEWAEENSR 412 LLHETDSAV 27 35 VLAGGFFLL 168 177 GMPEGDLVYV 441 450 LLQERGVAYI 469477 LMYSLVHNL 711 719 ALFDIESKV 663 671 MNDQVMFL 178 186 NYARTEDFF 227235 LYSDPADYF 624 632 TYSVSFDSL 334 348 TGNFSTQKVKMHIHS 459 473NYTLRVDCTPLMYSL 687 701 YRHVIYAPSSHNKYA 730 744 RQIYVAAFTVQAAAE

Example 4 Construction of Multi-Antigen Vaccine Constructs

In this Example, several strategies for expressing multiple antigensfrom single component DNA vaccine construct are described. Thesemulti-antigen DNA vaccine constructs share the same general plasmidbackbone as pPJV7563. Although the multi-antigen expression strategiesare described here in the context of a DNA vaccine, the principles willapply similarly in the context of viral vector genetic vaccines (such asadenovirus vectors). Unless otherwise specified, the genes included inthe multi-antigen constructs encode the human PSMA modified antigen(noted as PSMA), full length human PSCA (noted as PSCA), and the humanPSA cytosolic antigen (noted as PSA), as described in the examplesherein above.

Example 4A Dual Antigen Constructs

4A1. Construction of Dual Antigen Constructs Utilizing MultiplePromoters

General Strategy.

One strategy for creating multivalent nucleic acid vaccine constructs isto incorporate multiple independent promoters into a single plasmid(Huang, Y., Z. Chen, et al. (2008). “Design, construction, andcharacterization of a dual-promoter multigenic DNA vaccine directedagainst an HIV-1 subtype C/B’ recombinant.” J Acquir Immune Defic Syndr47(4): 403-411; Xu, K., Z. Y. Ling, et al. (2011). “Broad humoral andcellular immunity elicited by a bivalent DNA vaccine encoding HA and NPgenes from an H5N1 virus.” Viral Immunol 24(1): 45-56). The plasmid canbe engineered to carry multiple expression cassettes, each consisting ofa) a eukaryotic promoter for initiating RNA polymerase dependenttranscription, with or without an enhancer element, b) a gene encoding atarget antigen, and c) a transcription terminator sequence. Upondelivery of the plasmid to the transfected cell nucleus, transcriptionwill be initiated from each promoter, resulting in the production ofseparate mRNAs, each encoding one of the target antigens. The mRNAs willbe independently translated, thereby producing the desired antigens.

Plasmid 460 (PSMA/PSCA Dual Promoter).

Plasmid 460 was constructed using the techniques of site-directedmutagenesis, PCR, and restriction fragment insertion. First, a Kpn Irestriction site was introduced upstream of the CMV promoter in plasmid5259 using site-directed mutagenesis with MD5 and MD6 primers accordingto manufacturer's protocol (Quickchange kit, Agilent Technologies, SantaClara, Calif.). Second, an expression cassette consisting of a minimalCMV promoter, human PSMA, and rabbit B globulin transcription terminatorwas amplified by PCR from plasmid 5166 using primers that carried Kpn Irestriction sites (MD7 and MD8). The PCR amplicon was digested with KpnI and inserted into the newly introduced Kpn I site of calf intestinalalkaline phosphatase (CIP)-treated plasmid 5259.

4A2. Construction of Dual Antigen Constructs Utilizing 2A Peptides

General Strategy.

Multiple protein antigens can also be expressed from a single vectorthrough the use of viral 2A-like peptides (Szymczak, A. L. and D. A.Vignali (2005). “Development of 2A peptide-based strategies in thedesign of multicistronic vectors.” Expert Opin Biol Ther 5(5): 627-638;de Felipe, P., G. A. Luke, et al. (2006). “E unum pluribus: multipleproteins from a self-processing polyprotein.” Trends Biotechnol 24(2):68-75; Luke, G. A., P. de Felipe, et al. (2008). “Occurrence, functionand evolutionary origins of ‘2A-like’ sequences in virus genomes.” J GenVirol 89 (Pt 4): 1036-1042; Ibrahimi, A., G. Vande Velde, et al. (2009).“Highly efficient multicistronic lentiviral vectors with peptide 2Asequences.” Hum Gene Ther 20(8): 845-860; Kim, J. H., S. R. Lee, et al.(2011). “High cleavage efficiency of a 2A peptide derived from porcineteschovirus-1 in human cell lines, zebrafish and mice.” PLoS One 6(4):e18556). These peptides, also called cleavage cassettes or CHYSELs(cis-acting hydrolase elements), are approximately 20 amino acids longwith a highly conserved carboxy terminal D-V/I-EXNPGP motif (FIG. 2).The cassettes are rare in nature, most commonly found in viruses such asFoot-and-mouth disease virus (FMDV), Equine rhinitis A virus (ERAV),Encephalomyocarditis virus (EMCV), Porcine teschovirus (PTV), and Thoseaasigna virus (TAV) (Luke, G. A., P. de Felipe, et al. (2008).“Occurrence, function and evolutionary origins of ‘2A-like’ sequences invirus genomes.” J Gen Virol 89 (Pt 4): 1036-1042). With a 2A-basedmulti-antigen expression strategy, genes encoding multiple targetantigens can be linked together in a single open reading frame,separated by 2A cassettes. The entire open reading frame can be clonedinto a vector with a single promoter and terminator. Upon delivery ofthe genetic vaccine to a cell, mRNA encoding the multiple antigens willbe transcribed and translated as a single polyprotein. Duringtranslation of the 2A cassettes, ribosomes skip the bond between theC-terminal glycine and proline. The ribosomal skipping acts like acotranslational autocatalytic “cleavage” that releases upstream fromdownstream proteins. The incorporation of a 2A cassette between twoprotein antigens results in the addition of ˜20 amino acids onto theC-terminus of the upstream polypeptide and 1 amino acid (proline) to theN-terminus of downstream protein. In an adaptation of this methodology,protease cleavage sites can be incorporated at the N terminus of the 2Acassette such that ubiquitous proteases will cleave the cassette fromthe upstream protein (Fang, J., S. Yi, et al. (2007). “An antibodydelivery system for regulated expression of therapeutic levels ofmonoclonal antibodies in vivo.” Mol Ther 15(6): 1153-1159).

Plasmid 451 (PSMA-T2A-PSCA).

Plasmid 451 was constructed using the techniques of overlapping PCR andrestriction fragment exchange. First, the gene encoding human PSMA aminoacids 15-750 was amplified by PCR using plasmid 5166 as a template withprimers 119 and 117. The gene encoding full-length human PSCA wasamplified by PCR using plasmid 5259 as a template with primers 118 and120. PCR resulted in the addition of overlapping TAV 2A (T2A) sequencesat the 3′ end of PSMA and 5′ end of PSCA. The amplicons were mixedtogether and amplified by PCR with primers 119 and 120. ThePSMA-T2A-PSCA amplicon was digested with Nhe I and Bgl II and insertedinto similarly digested plasmid 5166. A glycine-serine linker wasincluded between PSMA and the T2A cassette to promote high cleavageefficiency.

Plasmid 454 (PSCA-F2A-PSMA).

Plasmid 454 was created using the techniques of PCR and restrictionfragment exchange. First, the gene encoding full-length human PSCA wasamplified by PCR using plasmid 5259 as a template with primers 42 and132. The amplicon was digested with BamH I and inserted into similarlydigested, CIP-treated plasmid 5300. A glycine-serine linker was includedbetween PSCA and the FMDV 2A (F2A) cassette to promote high cleavageefficiency.

Plasmid 5300 (PSA-F2A-PSMA)

Plasmid 5300 was constructed using the techniques of overlapping PCR andrestriction fragment exchange. First, the gene encoding PSA amino acids25-261 was amplified by PCR from plasmid 5297 with primers MD1 and MD2.The gene encoding human PSMA amino acids 15-750 was amplified by PCRfrom plasmid 5166 with primers MD3 and MD4. PCR resulted in the additionof overlapping F2A sequences at the 3′ end of PSA and 5′ end of PSMA.The amplicons were mixed together and extended by PCR. The PSA-F2A-PSMAamplicon was digested with Nhe I and Bgl II and inserted into similarlydigested plasmid pPJV7563.

4A3. Dual Antigen Constructs Utilizing Internal Ribosomal Entry Sites

General Strategy:

A third strategy for expressing multiple protein antigens from a singleplasmid or vector involves the use of an internal ribosomal entry site,or IRES. Internal ribosomal entry sites are RNA elements (FIG. 3) foundin the 5′ untranslated regions of certain RNA molecules (Bonnal, S., C.Boutonnet, et al. (2003). “IRESdb: the Internal Ribosome Entry Sitedatabase.” Nucleic Acids Res 31(1): 427-428). They attract eukaryoticribosomes to the RNA to facilitate translation of downstream openreading frames. Unlike normal cellular 7-methylguanosine cap-dependenttranslation, IRES-mediated translation can initiate at AUG codons farwithin an RNA molecule. The highly efficient process can be exploitedfor use in multi-cistronic expression vectors (Bochkov, Y. A. and A. C.Palmenberg (2006). “Translational efficiency of EMCV IRES in bicistronicvectors is dependent upon IRES sequence and gene location.”Biotechniques 41(3): 283-284, 286, 288). Typically, two transgenes areinserted into a vector between a promoter and transcription terminatoras two separate open reading frames separated by an IRES. Upon deliveryof the genetic vaccine to the cell, a single long transcript encodingboth transgenes will be transcribed. The first ORF will be translated inthe traditional cap-dependent manner, terminating at a stop codonupstream of the IRES. The second ORF will be translated in acap-independent manner using the IRES. In this way, two independentproteins can be produced from a single mRNA transcribed from a vectorwith a single expression cassette.

Plasmid 449 (PSMA-mIRES-PSCA).

Plasmid 449 was constructed using the techniques of overlapping PCR andrestriction fragment exchange. First, the gene encoding full lengthhuman PSCA was amplified by PCR from plasmid 5259 with primers 124 and123. The minimal EMCV IRES was amplified by PCR from pShuttle-IRES withprimers 101 and 125. The overlapping amplicons were mixed together andamplified by PCR with primers 101 and 123. The IRES-PSCA amplicon wasdigested with Bgl II and BamH I and inserted into Bgl II-digested,CIP-treated plasmid 5166. In order to fix a spontaneous mutation withinthe IRES, the IRES containing Avr II to Kpn I sequence was replaced withan equivalent fragment from pShuttle-IRES.

Plasmid 603 (PSCA-pIRES-PSMA).

Plasmid 603 was constructed using the techniques of PCR and seamlesscloning. The gene encoding full length human PSCA attached at its 3′ endto a preferred EMCV IRES was amplified from plasmid 455 by PCR withprimers SD546 and SD547. The gene encoding human PSMA amino acids 15-750was amplified by PCR from plasmid 5166 using primers SD548 and SD550.The two overlapping PCR amplicons were inserted into Nhe I and BglII-digested pPJV7563 by seamless cloning according to manufacturer'sinstructions (Invitrogen, Carlsbad, Calif.).

Plasmid 455 (PSCA-mIRES-PSA).

Plasmid 455 was constructed using the techniques of overlapping PCR andrestriction fragment exchange. First, the gene encoding human PSA aminoacids 25-261 was amplified by PCR from plasmid 5297 with primers 115 and114. The minimal EMCV IRES was amplified by PCR from pShuttle-IRES withprimers 101 and 116. The overlapping amplicons were mixed together andamplified by PCR with primers 101 and 114. The IRES-PSA amplicon wasdigested with Bgl II and BamH I and inserted into Bgl II-digested,CIP-treated plasmid 5259. In order to fix a spontaneous mutation withinthis clone, the Bgl II to BstE II sequence was replaced with anequivalent fragment from a fresh overlapping PCR reaction.

Example 4B Triple Antigen DNA Constructs

General Strategy.

The abilities of the dual antigen expression vectors to direct theexpression of PSMA, PSCA, and/or PSA were characterized in transfectedHEK293 cells (FIGS. 4, 5A, 5B, and 6). A number of dual antigenexpression cassettes, including PSA-F2A-PSMA, PSMA-mIRES-PSCA,PSMA-T2A-PSCA, PSA-T2A-PSCA, PSCA-F2A-PSMA, PSCA-pIRES-PSMA, andPSMA-mIRES-PSA, were selected for incorporation in various combinationsinto triple antigen expression vectors. In all cases, the vectors werebased on the parental pPJV7563 plasmid backbone. Four vectors (plasmids456, 457, 458, and 459) utilized a single full CMV promoter with arabbit B globulin transcription terminator to drive expression of allthree antigens. Two other vectors (plasmids 846 and 850) incorporated adual promoter strategy in combination with either an IRES or 2A to driveexpression of the three antigens. Vectors with multiple 2A cassetteswere engineered to carry different cassettes to minimize the likelihoodof recombination between the first and second cassette duringplasmid/vector amplification. Antigen expression was demonstrated byflow cytometry (FIGS. 7A and 7B) and western blotting (FIGS. 8A and 8B).

Plasmid 456 (PSA-F2A-PSMA-mIRES-PSCA).

Plasmid 456 was constructed by restriction fragment exchange. Plasmid5300 was digested with Nhe I and Hpa I and the ˜1.8 kb insert wasligated into similarly digested plasmid 449.

Plasmid 457 (PSA-F2A-PSMA-T2A-PSCA).

Plasmid 457 was constructed by restriction fragment exchange. Plasmid5300 was digested with Nhe I and Hpa I and the ˜1.8 kb insert wasligated into similarly digested plasmid 451.

Plasmid 458 (PSA-T2A-PSCA-F2A-PSMA).

Plasmid 458 was constructed using the techniques of PCR and restrictionfragment exchange. The gene encoding human PSA amino acids 25-261 wasamplified by PCR from plasmid 5297 with primers 119 and 139, resultingin the addition of a T2A sequence and Nhe I restriction site at the 3′end. The amplicon was digested with Nhe I and inserted into similarlydigested plasmid 454.

Plasmid 459 (PSCA-F2A-PSMA-mIRES-PSA).

Plasmid 459 was constructed by restriction fragment exchange. Plasmid454 was digested with Nhe I and Bgl II and the PSCA-F2A-PSMA containinginsert was ligated into similarly digested plasmid 455.

Plasmid 846 (CBA-PSA, CMV-PSCA-pIRES-PSMA).

Plasmid 846 was constructed using the techniques of PCR and seamlesscloning. First, an expression cassette was synthesized that consistedof 1) the promoter and 5′ untranslated region from the chicken betaactin (CBA) gene, 2) a hybrid chicken beta actin/rabbit beta globinintron, 3) the gene encoding human PSA amino acids 25-261, and 4) thebovine growth hormone terminator. This PSA expression cassette wasamplified by PCR from plasmid 796 with primers 3SalICBA and 5SalIBGH.The amplicon was cloned into the SalI site of plasmid 603 using aGeneArt Seamless Cloning and Assembly Kit (Invitrogen, Carlsbad,Calif.). Upon delivery of this plasmid into a cell, PSA expression willbe driven off the CBA promoter while PSCA and PSMA expression will bedriven off the CMV promoter.

Plasmid 850 (CBA-PSA, CMV-PSCA-F2A-PSMA).

Plasmid 850 was constructed using the techniques of PCR and seamlesscloning. First, the CBA promoter-driven PSA expression cassette wasamplified by PCR from plasmid 796 with primers 3SalICBA and 5SalIBGH.The amplicon was cloned into the SalI site of plasmid 454 using GeneArtSeamless Cloning. Upon delivery of this plasmid into a cell, PSAexpression will be driven off the CBA promoter while PSCA and PSMAexpression will be driven off the CMV promoter.

TABLE 20 List of Plasmids Expressing Multiple-Antigens 1^(st) Expression2^(nd) Expression 3^(rd) Plasmid Antigen strategy Antigen strategyAntigen ID # PSMA 5166 PSCA 5259 PSA 5297 PSMA 2 PSCA 460 promoters PSMAT2A PSCA 451 PSCA F2A PSMA 454 PSA F2A PSMA 5300 PSMA IRES PSCA 449 PSCAIRES PSMA 603 PSCA IRES PSA 455 PSA F2A PSMA mIRES PSCA 456 PSA F2A PSMAT2A PSCA 457 PSA T2A PSCA F2A PSMA 458 PSCA F2A PSMA mIRES PSA 459 PSA 2PSCA pIRES PSMA 796 promoters PSA 2 PSCA pIRES PSMA 846 promoters PSA 2PSCA F2A PSMA 850 promoters

TABLE 21 List of Primers Used in the Construction of the Multi-antigenPlasmids Primer Sequence (5′ to 3′) Strand  42 CGTTGACGCAAATGGGCGGTAGGSense 101 TCAGAGATCTGACCCCCTAACGTTACTGGC Sense 114TATAGGATCCTCAGGGGTTGGCCACGATG Antisense 115GAAAAACACGATGATAATATGGCCAGCATTGTGGGAGGCTGGGAGTG Sense 116CCACAATGCTGGCCATATTATCATCGTGTTTTTCAAAGGAAAACCACGT Antisense CC 117CATCTCCACAGGTCAATAATGAACCCCTACCTTCGGATCCGGCTACTTC Antisense ACTCAAAGTC118 GTTCATTATTGACCTGTGGAGATGTCGAAGAAAACCCAGGACCCGCAA SenseGCAAGGCTGTGCTGCTTGCCCTG 119 TTGCCTCTCACATCTCGTCAATCTCCGCGAGGAC Sense 120GATCTTTTGTACAATATGATCTTGTGGCAATGTCCC Antisense 123TATAGGATCCCTATAGCTGGCCGGGTCC Antisense 124CACGATGATAATATGGCCAGCAAGGCTGTGCTGCTTGCC Sense 125CACAGCCTTGCTGGCCATATTATCATCGTGTTTTTCAAAGGAAAACCAC Antisense GTCC 132TATAGGATCCTAGCTGGCCGGGTCCCCAGAG Antisense 139ATATGCTAGCGGGTCCTGGGTTTTCTTCGACATCTCCACAGGTCAATAA AntisenseTGAACCCCTACCTTCGGATCCGGGG TTGGCCACGATGGTGTCC SD546CTGTGACGAACATGGCTAGCAAGG Sense SD547 ATTATCATCGTGTTTTTCAAAGGAAAACCAntisense SD548 AAACACGATGATAATATGGCCACAACCATGGCGCGCCGCCCGC Sense SD550TTTTGTTAGGGCCCAGATCTTTAGGC Antisense MD1 GACGAACATGGCTAGCATTGTGGGAGGCTGSense MD2 CCACATCGCCTGCCAGTTTCAGCAGATCAAAGTTCAGGGTCTGGGATC AntisenseCGGGGTTGGCCACGATGGTGTC MD3GATCTGCTGAAACTGGCAGGCGATGTGGAAAGCAACCCAGGCCCAATG SenseGCAAGCGCGCGCCGCCCGCGCTG MD4 GTTAGGGCCCAGATCTTTAGGCTACTTCACTCAAAGTCAntisense MD5 CTTGTATTACTGTTTATGTAAGCAGACAGGGTACCAATATTGGCTATTG SenseGCCATTGCATAC MD6 GTATGCAATGGCCAATAGCCAATATTGGTACCCTGTCTGCTTACATAAAAntisense CAGTAATACAAG MD7 CATGCATGGGTACCAATCTTCCGAGTGAGAGACACAAAAAATTCCSense MD8 GATCGATCGGTACCCTGCAGGTCGAGCACCAAAATCAACGGG Antisense 5SalIBGHGTTTATGTAAGCAGACAGGTCGACCCATAGAGCCCACCGCATCCCCAGC Antisense 3SalICBATGGCCAATAGCCAATATTGTCGACTGGGTCGAGGTGAGCCCCACGTTC Sense TG

Example 4C Triple Antigen Adenovirus Constructs

General Strategy.

As with DNA plasmids, viral vaccine vectors can be engineered to delivermultiple prostate cancer antigens. The three multi-antigen expressionstrategies described above for DNA vaccines—dual promoters, 2A peptides,and internal ribosome entry sites—were incorporated in variouscombinations to create triple antigen adenovirus vectors. Briefly, themulti-antigen expression cassettes were cloned into a pShuttle-CMVplasmid modified to carry two copies of the tetracycline operatorsequence (TetO2). Recombinant adenovirus serotype 5 vectors were createdusing the AdEasy Vector System according to manufacturer's protocols(Agilent Technologies, Inc., Santa Clara, Calif.). Viruses wereamplified in HEK293 cells and purified by double cesium chloride bandingaccording to standard protocols. Prior to in vivo studies, viral stockswere thoroughly characterized for viral particle concentration,infectivity titer, sterility, endotoxin, genomic and transgeneintegrity, transgene identity and expression.

Adenovirus-733 (PSA-F2A-PSMA-T2A-PSCA).

Ad-733 is the viral equivalent of plasmid 457. Expression of the threeantigens is driven off a single CMV promoter with a tetracyclineoperator for repressing transgene expression during large scaleproduction in Tet repressor expressing HEK293 lines. Multi-antigenexpression strategies include two different 2A sequences.

Adenovirus-734 (PSA-T2A-PSCA-F2A-PSMA).

Ad-734 is the viral equivalent of plasmid 458. Expression of the threeantigens is driven off a single CMV promoter with a tetracyclineoperator for repressing transgene expression during large scaleproduction in Tet repressor expressing HEK293 lines. Multi-antigenexpression strategies include two different 2A sequences.

Adenovirus-735 (PSCA-F2A-PSMA-mIRES-PSA).

Ad-735 is the viral equivalent of plasmid 459. Expression of the threeantigens is driven off a single CMV promoter with a tetracyclineoperator for repressing transgene expression during large scaleproduction in Tet repressor expressing HEK293 lines. Multi-antigenexpression strategies include a 2A sequence and an IRES.

Adenovirus-796 (CBA-PSA, CMV-PSCA-pIRES-PSMA).

Ad-796 is the viral equivalent of plasmid 846. Expression of PSA isdriven off the chicken beta actin promoter while PSCA and PSMAexpression is driven off the CMV-TetO2 promoter. Multi-antigenexpression strategies include two promoters and an IRES.

Adenovirus-809 (CBA-PSA, CMV-PSCA-F2A-PSMA).

Ad-809 is the viral equivalent of plasmid 850. Expression of PSA isdriven off the chicken beta actin promoter while PSCA and PSMAexpression is driven off the CMV-TetO2 promoter. Multi-antigenexpression strategies include two promoters and a 2A sequence.

Example 5 Immunogenicity of Triple Antigen DNA Vaccines

Example 5 illustrates the capability of triple antigen nucleic acidvaccine constructs expressing PSMA, PSCA and PSA to elicitantigen-specific T and B cell responses to all three encoded prostateantigens.

Cellular Immune Response Study.

Immunogenicity of triple antigen constructs containing PSMA, PSCA andPSA, as described in Example 5, was studied in C57BL/6 mice according tothe procedure described below.

Female C57BL/6 mice were primed on day 0 and boosted on days 14, 28 and49 with DNA vaccine constructs encoding human-PSMA, PSCA and PSAantigens by PMED administration. In total, four different triple antigenvaccination strategies were evaluated, which included three DNA vaccinesthat co-expressed the target proteins and one co-formulation approach.For co-expression, single DNA plasmids encoding all three prostateantigens linked by 2A peptides or internal ribosome entry sites (IRES)were used as follows: PSA-F2A-PSMA-T2A-PSCA (plasmid ID#457),PSA-T2A-PSCA-F2A-PSMA (plasmid ID#458) and PSCA-F2A-PSMA-IRES-PSA(plasmid ID#459). For the co-formulation approach, three different DNAplasmids, each individually encoding PSMA, PSCA or PSA, wereco-formulated onto a single gold particle for PMED delivery. With theexception of co-formulation, the DNA elements that control co-expression(2A and IRES) differ in length, transgene expression efficiency and thepresence of foreign genetic material attached to the target transgenes.As controls, C57BL/6 mice were vaccinated with DNA expressing a singleprostate antigen, either PSMA, PSCA or PSA. For the co-expressed tripleor single antigen DNA vaccines, a dose 2 μg of DNA vaccine plasmid wasgiven per PMED administration, whereas 1 μg of each of the co-formulatedtriple antigen DNA vaccines (a total of 3 μg) was administered per PMEDadministration. Cellular immune responses against the triple and singleantigen vaccines were measured by collecting the spleens from eachanimal on day 56, seven days after the final PMED vaccination.Splenocytes were isolated and subjected to an IFN-γ ELISPOT assay tomeasure the PSMA, PSCA and PSA-specific T cell responses. Briefly, 2×10⁵splenocytes from individual animals were plated per well with 5×10⁴ perwell of TRAMP-C2 (transgenic adenocarcinoma mouse prostate) cells stablyexpressing a single human prostate antigen or PSMA, PSCA and PSAtogether, or with individual or pools of human PSMA, PSCA andPSA-specific peptides at 10 μg/ml (see Table 22 for peptides and peptidepool composition), or medium alone as a control. Each condition wasperformed in triplicate. The plates were incubated for 20 h at 37° C.and 5% CO₂, washed and developed after incubation as per themanufacturer's instructions. The number of IFN-γ spot forming cells(SFC) was counted by a Cellular Technology Ltd. (CTL) reader. Theresults are presented in FIGS. 9 and 10, which show the average numberof PSMA, PSCA or PSA-specific SFCs+/−the standard deviation of five miceper group, normalized to 1×10⁶ splenocytes.

TABLE 22 The 15mer PSMA, PSCA and PSA peptides that were tested in theELISPOT assay. The amino acid position of the N and C-terminal end ofeach peptide is indicated. Prostate antigen Peptides Tested individuallyor pool PSMA 577-591 Individual PSMA 589-603 Individual PSMA 601-615Individual PSMA 629-643 Individual PSMA 641-655 Individual PSMA 77-91Pool 1  91-111 153-167 229-243 365-379 PSMA 401-415 Pool 2 429-443521-535 613-627 PSMA 657-671 Pool 3 685-699 701-715 733-747 PSCA 25-39Individual PSA 65-79 Individual PSA 73-87 Individual

Antibody Response Study.

Antibody responses against the triple and single antigen vaccines weremeasured by collecting the serum from each animal on day 56, seven daysafter the final PMED vaccination. Serum was subjected to enzyme-linkedimmunosorbent assays (ELISA) to determine the anti-PSMA and anti-PSCAantibody titers. In brief, ELISA plates were coated with 1 μg/ml ofhuman PSMA or PSCA and incubated overnight at 4° C. Plates were thenblocked and incubated at RT for 1 h with 1% bovine serum albumin (BSA).Each serum sample was serially diluted in duplicate starting at a 1:100dilution and incubated for 1 h at RT. After washing, ahorseradish-peroxidise (HRP)-conjugated goat anti-mouse polyclonal IgGantibody was incubated at RT for 1 h. After washing, the TMB PeroxidaseEIA-Substrate was incubated at RT for 30 min. The colorimetric reactionwas stopped by addition 1N sulfuric acid and the absorbance then read at450 nm. Titration curves were plotted for each serum sample (sampledilution versus absorbance). The serum titer (subsequently transformedinto reciprocal titer) was then taken as the most dilute serum sampletested with an optical density (OD) value of above the lower limit ofdetection (LLOD; background plus 3 standard deviations) or the serumdilution calculated to achieve an OD value of 1.0. The results arepresented in FIGS. 11 and 12, which show the average titers+/−thestandard deviation of five mice per group.

Serum was also subjected to a fluorescence-activated cell sorting (FACS)assay to measure antibody binding to either human PSMA or PSCA expressedon the cell surface of appropriate cell lines, thus determining whetherantibodies generated by the multi-antigen vaccines were capable ofrecognizing native PSMA and PSCA conformations, respectively. LNCaP(human prostate adenocarcinoma) cells were utilized to measure antibodybinding to native PSMA. PC3 (human prostate cancer) cells served as acontrol in the FACS assay, as these cells do not express human PSMA.MIA-PaCa-2 (human pancreatic carcinoma) cells transduced with anadenovirus expressing human PSCA (Ad-PSCA) were utilized to measureantibody binding to native PSCA. Untransduced MIA-PaCa-2 cells served asthe control. In brief, to measure anti-PSMA antibody binding, 2×10⁵LNCaP or PC3 cells were incubated with a 1:100 dilution of mouse serumor 15 μg/ml of the control mouse-anti-human PSMA monoclonal antibody(mAb) (clone J591-A) for 20 min at 4° C. To measure anti-PSCA antibodybinding, 2×10⁵ Ad-PSCA transduced and untransduced MIA-PaCa-2 cells wereincubated with a 1:30 dilution of mouse serum or 4 μg/ml of the controlmouse anti-human PSCA mAb (clone 7F5) for 20 min at 4° C. Subsequently,cells were washed and incubated with a secondary Phycoerythrin(PE)-conjugated goat-anti-mouse IgG antibody and a live/dead dye for anadditional 20 min at 4° C. After the incubation, cells were washed andresuspended in 1.5% paraformaldehyde, and 10,000 live cells wereacquired on a FACS Canto II. The results are presented in FIGS. 13 and14, which show the average fold change in mean fluorescence intensity(MFI) of the mouse serum over the secondary anti-mouse antibodyalone+/−the standard deviation of five mice per group. Antibody titerswere not measured because PSA was expressed as a cytoplasmic protein bythe multi-antigen vaccines investigated in this study.

Results:

FIGS. 9A-9D show the results of a representative study that evaluatesthe cellular immune responses of the triple antigen vaccines by IFN-γELISPOT assay. Briefly, 5 mice per group were primed on day 0 andboosted with PMED on days 14, 28 and 49. On day 56, seven days after thelast PMED vaccination, recognition of the endogenous prostate antigenswas assessed by examining T cell responses to (A) TRAMP C2-PSMA, (B)TRAMP C2-PSCA, (C) TRAMP C2-PSA, and (D) TRAMP C2-PSMA-PSA-PSCA cells byIFN-γ ELISPOT assay. The TRAMP C2 cells served as a background controlfor the assay. For IFN-γ T cell responses to endogenous PSMA, asignificant response to TRAMP C2-PSMA was observed following the singlePSMA PMED vaccination, which was consistent with responses seen in otherstudies. Similar PSMA-specific IFN-γ T cell response to TRAMP C2-PSMAwas detected following the triple antigen vaccinations. In contrast,complete ablation of the response was observed following co-formulatedPSMA, PSCA and PSA vaccination (* indicates p<0.05 by two-way ANOVA).For IFN-γ T cell responses to endogenous PSCA, no significant differencein response to the TRAMP C2-PSCA cells was observed when comparing thesingle PSCA vaccine to the four different triple antigen vaccines. ForIFN-γ T cell responses to endogenous PSA, a significant decrease in theresponse magnitude to TRAMP C2-PSA was detected when comparing theimmunogenicity of the single PSA vaccine to eitherPSCA-F2A-PSMA-IRES-PSA (*** indicates p<0.001 by two-way ANOVA) or theco-formulated vaccine (* indicates p<0.05 by two-way ANOVA). Whenexamining the response to TRAMP C2-PSMA-PSCA-PSA, the highest magnitudeIFN-γ T cell response was observed following the PSA-F2A-PSMA-T2A-PSCAvaccine. Taken together, these data demonstrate recognition ofendogenous PSMA, PSCA and PSA and generation of antigen-specific T cellresponses to all three prostate antigens using a co-expression DNAvaccination strategy, especially with the PSA-F2A-PSMA-T2A-PSCA vaccineconstruct. However, the co-formulation DNA vaccination strategy resultedin a loss of antigen-specific IFN-γ T cell responses to PSMA and PSA.

FIGS. 10A-10D show the results of a representative study that evaluatesthe immunogenicity of the triple antigen vaccines by IFN-γ ELISPOTassay. Briefly, 5 mice per group were primed on day 0 and boosted withPMED on days 14, 28 and 49. On day 56, T cell responses to (A) PSMApeptides, (B) PSMA peptide pools, (C) PSCA peptides and (D) PSA peptides(see Table 22) were assessed by IFN-γ ELISPOT assay. Medium alone servedas a background control for the assay. For IFN-γ T cell responses toboth the individual and pools of PSMA peptides, compared to the singlePSMA vaccine, the highest magnitude response was observed followingadministration of the PSA-F2A-PSMA-T2A-PSCA triple antigen vaccine.Similarly, the highest magnitude IFN-γ T cell response to PSCA andPSA-specific peptides was detected following administration of thePSA-F2A-PSMA-T2A-PSCA vaccine. The co-formulated PSMA, PSCA and PSAvaccine resulted in low to no T cell responses to the PSMA-specificpeptides and low magnitude responses to the PSCA and PSA-specificpeptides. These data also demonstrate generation of T cell responses toPSMA, PSCA and PSA when co-expressed from the same vaccine construct.There was consistent and robust IFN-γ T cell responses to all threeprostate antigens following PSA-F2A-PSMA-T2A-PSCA vaccination, andsignificant decreases in the magnitude of IFN-γ T cell responses to theprostate antigens following PSA-T2A-PSCA-F2A-PSMA,PSMA-F2A-PSMA-IRES-PSA and co-formulated PSMA, PSCA and PSAvaccinations.

FIG. 11 shows the results of a representative study that evaluates theimmunogenicity of the triple antigen vaccines by anti-PSMA antibodytiters. Briefly, 5 mice per group were primed on day 0 and boosted withPMED on days 14, 28 and 49. On day 56, serum anti-PSMA antibody titerswere assessed by ELISA. All animals vaccinated with PSMA generatedsignificant anti-PSMA antibody titers. There were no significantdifferences between titers, although vaccination withPSA-F2A-PSMA-T2A-PSCA resulted in slightly lower titers compared to theother groups vaccinated with PSMA. These data demonstrate the generationof anti-PSMA-specific antibodies following triple antigen vaccination,using both co-expression and co-formulation vaccine strategies.

FIG. 12 shows the results of a representative study that evaluates theimmunogenicity of the triple antigen vaccines by anti-PSCA antibodytiters. Briefly, 5 mice per group were primed on day 0 and boosted withPMED on days 14, 28 and 49. On day 56, serum anti-PSCA antibody titerswere assessed by ELISA. Antibody titers were detected in mice vaccinatedwith PSCA alone and co-formulated PSMA, PSCA and PSA. These resultsindicate that co-formulation of PSMA, PSCA and PSA elicits a detectableanti-PSCA antibody titer compared to the co-expressed DNA vaccinationstrategies.

FIG. 13 shows the results of a representative study that evaluates theimmunogenicity of the triple antigen vaccines by anti-PSMA antibodycell-surface binding. Briefly, 5 mice per group were primed on day 0 andboosted with PMED on days 14, 28 and 49. On day 56, recognition ofcell-surface native PSMA was assessed by serum antibody binding to LNCaPand PC3 cells. The PC3 cells served as a background control for theassay. PSA-F2A-PSMA-T2A-PSCA vaccination resulted in anti-PSMAantibodies with a significantly lower binding capacity to LNCaP cellscompared to mice vaccinated with PSA-T2A-PSCA-F2A-PSMA and PSMA alone (*indicates p-value <0.05 by one-way ANOVA). All other PSMA vaccinatedgroups showed no significant difference in anti-PSMA antibody binding.The fold-change over secondary antibody alone for the J591-A mAb was45.3 (data not shown). Overall, these data demonstrate generation ofanti-PSMA-specific antibodies that recognize native PSMA followingtriple antigen vaccination, using both co-expression and co-formulationDNA vaccination strategies.

FIG. 14 shows the results of a representative study that evaluates theimmunogenicity of the triple antigen vaccines by anti-PSCA antibodycell-surface binding. Briefly, 5 mice per group were primed on day 0 andboosted with PMED on days 14, 28 and 49. On day 56, recognition ofcell-surface native PSCA was assessed by serum antibody binding toAd-PSCA transduced and untransduced MIA-PaCa-2 cells. The untransduced,parental cells served as a background control for the assay. With theexception of the single PSMA and single PSA cyto vaccines, all vaccineregimens with PSCA resulted in significant anti-PSCA antibody binding toAd-PSCA transduced MIA-PaCa-2 cells compared to the parental cells.There were no significant differences in the anti-PSCA antibody bindingto Ad-PSCA transduced MIA-PaCa-2 cells between the PSCA-vaccinatedgroups (one-way ANOVA, p-value >0.05). The fold change over secondaryantibody alone for the 7F5 mAb was 18.7 (data not shown). Overall, thesedata demonstrate the generation of anti-PSCA-specific antibodies thatrecognize native PSCA following triple antigen vaccination, using bothco-expression and co-formulation DNA vaccination strategies.

Example 6 Immunogenicity of Dual Antigen Vaccines

The following examples are provided to illustrate the capability of dualantigen vaccines expressing two prostate antigens to elicitantigen-specific T and B cell responses to the two encoded prostateantigens.

6A. Immunogenicity of Dual Antigen Vaccines Containing PSMA and PSCA inC57BL/6:

Study Procedure.

Cellular Immune Response Study. Female C57BL/6 mice were primed on day 0and boosted on days 14, 28, 42 and 70 with human PSMA and PSCAexpressing DNA by PMED epidermal injection. In total, five differentdual antigen DNA vaccination strategies were evaluated, which includedfour DNA vaccines that co-expressed the antigens and one co-formulationapproach. For co-expression, single DNA vaccine plasmids encoding twoprostate antigens, PSMA and PSCA, linked by a dual promoter, 2A peptidesor IRES were administered. These included PSMA-PSCA dual promoter(plasmid ID#460), PSMA-T2A-PSCA (plasmid ID#451), PSCA-F2A-PSMA (plasmidID#454) and PSCA-IRES-PSMA (plasmid ID#603). For co-formulation, twodifferent DNA plasmids, each individually encoding PSMA and PSCA, wereco-formulated onto a single gold particle for PMED delivery. With theexception of co-formulation, the DNA elements that control co-expression(dual promoter, 2A and IRES) differ in length, transgene expressionefficiency and the presence of foreign genetic material attached to thetarget transgenes. As controls, C57BL/6 mice were vaccinated with DNAexpressing a single prostate antigen, PSMA or PSCA. For the co-expresseddual or single antigen DNA vaccines, a total dose of 2 μg of DNA vaccinewas given per PMED administration, whereas 2 μg of each DNA vaccineplasmid (total of 4 μg of DNA per administration) was given for theco-formulation. Cellular immune responses of the dual and single antigenvaccines were measured by collecting the spleens from each animal on day77, seven days after the final PMED vaccination. Splenocytes wereisolated and subjected to an IFN-γ ELISPOT assay to measure the PSMA andPSCA-specific T cell responses. Briefly, 2×10⁵ splenocytes fromindividual animals were plated per well with 5×10⁴ per well of TRAMP-C2cells expressing a single endogenous human prostate antigen or PSMA,PSCA and PSA together, or with individual or pools of human PSMA andPSCA-specific peptides at 10 μg/ml (see Table 22 for peptides andpeptide pool composition), or medium alone as a control. Each conditionwas performed in triplicate. The plates were incubated for 20 h at 37°C. and 5% CO₂, washed and developed after incubation as per themanufacturer's instructions. The number of IFN-γ SFC was counted by aCTL reader. The results are presented in FIGS. 15 and 16, which show theaverage number of PSMA or PSCA-specific SFCs+/−the standard deviation offive mice per group, normalized to 1×10⁶ splenocytes.

Antibody Response Study.

Antibody responses against the dual and single antigen vaccines weremeasured by collecting the serum from each animal on day 77, seven daysafter the final PMED vaccination. The anti-PSMA and anti-PSCA antibodytiters in the serum was determined using ELISA as described in Example5. The results are presented in FIGS. 17 and 18, which show the averagetiters+/−the standard deviation of five mice per group.

Serum was also subjected to a FACS assay to measure antibody binding toeither human PSMA or PSCA expressed on the cell surface of appropriatecell lines, thus determining whether antibodies generated by themulti-antigen vaccines were capable of recognizing native PSMA and PSCAconformations, respectively. Antibody binding to cell-surface native PSAwas not measured because PSA was expressed as a cytoplasmic protein bythe multi-antigen vaccines investigated in this study. The FACS assaywas conducted according to procedure as described in Example 5. Theresults presented in FIGS. 19 and 20, show the average fold-change inMFI of the mouse serum over the secondary anti-mouse antibodyalone+/−the standard deviation of five mice per group. Antibody titersand binding to cell-surface native PSA were not measured because PSA wasexpressed as a cytoplasmic protein by the multi-antigen vaccinesinvestigated in this study.

Results.

FIGS. 15A-15C show the results of a representative study that evaluatesthe immunogenicity of the dual antigen vaccines by IFN-γ ELISPOT assay.Briefly, 5 mice per group were primed on day 0 and boosted with PMED ondays 14, 28, 42 and 70. On day 77, recognition of endogenous PSMA andPSCA was assessed by examining T cell responses to (A) TRAMP C2-PSMA,(B) TRAMP C2-PSCA and (C) TRAMP C2-PSMA-PSA-PSCA cells by IFN-γ ELISPOTassay. The TRAMP C2 cells served as a background control for the assay.For IFN-γ T cell responses to endogenous PSMA, the magnitude of theresponse TRAMP C2-PSMA was significantly decreased following vaccinationwith PSMA-PSCA dual promoter, PSMA-T2A-PSCA, PSCA-IRES-PSMA andco-formulated PSMA PSCA compared to vaccination with PSMA alone (** and*** indicate p-values <0.01 and <0.001, respectively, by two-way ANOVA).However, the PSCA-F2A-PSMA vaccine construct elicited a similarmagnitude IFN-γ T cell response to the TRAMP C2-PSMA cells as the singlePSMA vaccine. For IFN-γ T cell responses to endogenous PSCA,significantly increased responses were observed following vaccinationwith several of the dual antigen vaccines, including PSMA-PSCA dualpromoter, PSCA-F2A-PSMA, PSCA-IRES-PSMA and co-formulated PSMA PSCAcompared to the PSCA vaccine alone (*, ** and *** indicate p-values of<0.05, 0.01 and 0.001, respectively, by two-way ANOVA). ThePSCA-T2A-PSMA vaccine construct elicited a similar magnitude IFN-γ Tcell response to the TRAMP C2-PSCA cells as the single PSCA vaccine.Comparing the IFN-γ T cell responses to TRAMP C2-PSMA-PSA-PSCA, therewere no significant differences between the groups vaccinated withdifferent dual antigen vaccines. Taken together, these data demonstrategeneration of PSMA and PSCA-specific T cell responses following dualantigen vaccination, using both co-expression and co-formulation DNAvaccination strategies.

FIGS. 16A-16C show the results of a representative study that evaluatesthe immunogenicity of the dual antigen vaccines by IFN-γ ELISPOT assay.Briefly, 5 mice per group were primed on day 0 and boosted with PMED ondays 14, 28, 42 and 70. On day 77, T cell responses to (A) PSMApeptides, (B) PSMA peptide pools and (C) PSCA peptides (see Table 22)were assessed by IFN-γ ELISPOT assay. Medium alone served as abackground control for the assay. For IFN-γ T cell responses to both theindividual and pools of PSMA peptides, the highest magnitude responsescompared to the single PSMA vaccine were observed following thePSMA-T2A-PSCA and PSCA-F2A-PSMA dual antigen vaccinations. A significantreduction in the IFN-γ T cell response to the individual PSMA peptideswas observed following vaccination with PSMA-PSCA dual promoter,PSCA-IRES-PSMA and co-formulated PSMA PSCA. The IFN-γ T cell response tothe PSCA-specific peptide was similar between the groups vaccinated withthe different dual antigen vaccines. These data also demonstrategeneration of T cell responses to both PSMA and PSCA when co-expressedon the same DNA vaccine construct, or delivered as a co-formulation.

FIG. 17 shows the results of a representative study that evaluates theimmunogenicity of the dual antigen vaccines by anti-PSMA antibodytiters. Briefly, 5 mice per group were primed on day 0 and boosted withPMED on days 14, 28, 42 and 70. On day 77, serum anti-PSMA antibodytiters were assessed by ELISA. All animals vaccinated with PSMAgenerated significant anti-PSMA antibody titers. Mice vaccinated withthe dual vaccine construct, PSCA-F2A-PSMA, and the single PSMA vaccinegenerated significantly higher antibody titers compared to all othergroups of mice vaccinated with PSMA (one-way ANOVA, p-value <0.05).Vaccination with PSMA-PSCA dual promoter and co-formulated PSMA and PSCAresulted in higher antibody titers compared to mice that received thePSMA-T2A-PSCA vaccine. Taken together, these data demonstrate generationof anti-PSMA-specific antibodies following dual antigen DNA vaccinationwith PSMA and PSCA, using both co-expression and co-formulationvaccination strategies.

FIG. 18 shows the results of a representative study that evaluates theimmunogenicity of the dual antigen vaccines by anti-PSCA antibodytiters. Briefly, 5 mice per group were primed on day 0 and boosted withPMED on days 14, 28, 42 and 70. On day 77, serum anti-PSCA antibodytiters were assessed by ELISA. Mice vaccinated with the co-formulatedPSMA and PSCA, and the single PSCA vaccine generated significantlyhigher antibody titers compared to all other groups of mice vaccinatedwith PSCA (one-way ANOVA). Vaccination with PSMA-PSCA dual promoterresulted in higher antibody titers compared to vaccination withPSMA-T2A-PSCA, PSCA-F2A-PSMA and PSCA-IRES-PSMA. These results indicatethat co-expression or co-formulation of PSMA and PSCA elicits anti-PSCAantibodies.

FIG. 19 shows the results of a representative study that evaluates theimmunogenicity of the dual antigen vaccines by anti-PSMA antibodycell-surface binding. Briefly, 5 mice per group were primed on day 0 andboosted with PMED on days 14, 28, 42 and 70. On day 77, recognition ofcell-surface native PSMA was assessed by serum antibody binding to LNCaPand PC3 cells. The PC3 cells served as a background control for theassay. With the exception of the single PSCA vaccine, all vaccineregimens with PSMA resulted in significant anti-PSMA antibody binding toLNCaP cells compared to the control PC3 cells. There were no significantdifferences in the anti-PSMA antibody binding to LNCaP cells between thePSMA-vaccinated groups (one-way ANOVA, p-value >0.05). The fold changeover secondary antibody alone for the J591-A mAb was 45.3 (data notshown). These data demonstrate generation of anti-PSMA-specificantibodies that recognized native PSMA following dual antigen DNAvaccination, using both co-expression and co-formulation vaccinationstrategies.

FIG. 20 shows the results of a representative study that evaluates theimmunogenicity of the dual antigen vaccines by anti-PSCA antibodycell-surface binding. Briefly, 5 mice per group were primed on day 0 andboosted with PMED on days 14, 28, 42 and 70. On day 77, recognition ofcell-surface native PSCA was assessed by serum antibody binding toAd-PSCA transduced and untransduced MIA-PaCa-2 cells. The untransduced,parental cells served as a background control for the assay. With theexception of the single PSMA vaccine, all vaccine regimens with PSCAresulted in significant anti-PSCA antibody binding to Ad-PSCA transducedMIA-PaCa-2 cells compared to the control cells. There were nosignificant differences in the anti-PSCA antibody binding to Ad-PSCAtransduced MIA-PaCa-2 cells between the PSCA-vaccinated groups (one-wayANOVA, p-value >0.05). The fold change over secondary antibody alone forthe 7F5 mAb was 18.7 (data not shown). Overall, these data demonstrategeneration of anti-PSCA-specific antibodies that recognized native PSCAfollowing dual antigen DNA vaccination, using both co-expression andco-formulation vaccination strategies.

6B. Immunogenicity of Dual Antigen Vaccines Containing Either PSMA andPSA or PSCA and PSA in C57BL/6

Study Procedure.

Cellular Immune Response Study.

Female C57BL/6 mice were primed on day 0 and boosted on days 14 and 28with human PSMA, PSCA and PSA expressing DNA by PMED epidermalinjection. In total, four different dual antigen vaccines strategieswere evaluated, which included two co-expression approaches and twoco-formulation strategies. For co-expression, a single DNA plasmidencoding two prostate antigens, PSMA and PSA linked a 2A peptide(plasmid ID#5300) or PSCA and PSA linked by IRES (plasmid ID#455) wereadministered. For co-formulation, plasmids individually encoding PSMA,PSCA or PSA were co-formulated onto a single gold particle for PMEDdelivery. Specifically, these included PSMA and PSA co-formulated andPSCA and PSA co-formulated. As controls, C57BL/6 mice were vaccinatedwith DNA expressing a single prostate antigen, PSMA, PSCA or PSA. Forthe co-expressed dual or single antigen vaccines, a dose 2 μg of DNA wasgiven per PMED administration, whereas 2 μg of each DNA vaccine plasmid(total of 4 μg of DNA per administration) was given for theco-formulation. Cellular immune responses of the dual and single antigenvaccines were measured by collecting the spleens from each animal on day35. Splenocytes were isolated and subjected to an IFN-γ ELISPOT assay tomeasure the PSMA, PSCA and PSA-specific T cell responses. Briefly, 2×10⁵splenocytes from individual animals were plated per well with 5×10⁴ perwell of TRAMP-C2 cells expressing a single endogenous human prostateantigen or PSMA, PSCA and PSA together, or with individual or pools ofhuman PSMA, PSCA and PSA-specific peptides at 10 μg/ml (see Table 22 forpeptides and peptide pool composition), or medium alone as a control.Each condition was performed in triplicate. The plates were incubatedfor 20 h at 37° C. and 5% CO₂, washed and developed after incubation asper manufacturer's instructions. The number of IFN-γ SFC was counted bya CTL reader. The results are presented in FIGS. 21 and 22, which showthe average number of PSMA, PSCA and PSA-specific SFCs+/−the standarddeviation of five mice per group, normalized to 1×10⁶ splenocytes.

Antibody Response Study.

Female C57BL/6 mice were primed on day 0 and boosted on days 14, 28 and49 with human PSMA, PSCA and PSA expressing DNA by PMED. Antibodyresponses against the dual and single antigen vaccines were measured bycollecting the serum from each animal on day 56, seven days after thefinal PMED vaccination. The anti-PSMA and anti-PSCA antibody titers inthe serum was determined using ELISA assay as described in Example 5.The results are presented in FIGS. 23 and 24, which show the averagetiters+/−the standard deviation of five mice per group.

Serum was also subjected to a FACS assay to measure antibody binding toeither human PSMA or PSCA expressed on the cell surface of appropriatecell lines, thus determining whether antibodies generated by themulti-antigen vaccines were capable of recognizing native PSMA and PSCAconformations, respectively. Antibody binding to cell-surface native PSAwas not measured because PSA was expressed as a cytoplasmic protein bythe multi-antigen vaccines investigated in this study. The FACS assaywas conducted according to the procedure as described in Example 5. Theresults are presented in FIGS. 25 and 26, which show the average foldchange in MFI of the mouse serum over the secondary anti-mouse antibodyalone+/−the standard deviation of five mice per group. Antibody titersand binding to cell-surface native PSA were not measured because PSA wasexpressed as a cytoplasmic protein by the multi-antigen vaccinesinvestigated in this study.

Results.

FIGS. 21A-21D show the results of a representative study that evaluatesthe immunogenicity of the dual antigen vaccines by IFN-γ ELISPOT assay.Briefly, 5 mice per group were primed on day 0 and boosted with PMED ondays 14 and 28. On day 35, recognition of endogenous PSMA, PSCA and PSAwas assessed by examining T cell responses to (A) TRAMP C2-PSMA, (B)TRAMP C2-PSCA, (C) TRAMP C2-PSA and (D) TRAMP C2-PSMA-PSA-PSCA cells byIFN-γ ELISPOT assay. The TRAMP C2 cells served as a background controlfor the assay. For IFN-γ T cell responses to endogenously expressed PSMAon cells, no significant differences were observed between responses toTRAMP C2-PSMA following vaccination with dual antigens containing PSMA(PSA-F2A-PSMA and co-formulated PSMA and PSA) and PSMA alone. Likewise,for IFN-γ T cell responses to endogenous PSCA, there were no observeddifferences in response magnitude to TRAMP C2-PSCA between the dualPSCA-IRES-PSA and co-formulated PSCA and PSA vaccines compared to thesingle PSCA vaccine. For IFN-γ T cell responses to endogenous PSA, asignificant increase in the response magnitude to TRAMP C2-PSA wasdetected when comparing the immunogenicity of the single PSA vaccine toeither PSA-F2A-PSMA (*** indicates p<0.001 by two-way ANOVA) andco-formulated PSMA and PSA (** indicates p<0.01 by two-way ANOVA). Therewere no observed differences in response magnitude to TRAMP C2-PSA whencomparing animals that received the dual PSCA and PSA vaccines to thesingle PSA vaccine. When examining the IFN-γ T cell response to TRAMPC2-PSMA-PSA-PSCA, there were no significant differences in the responsebetween the groups vaccinated with different dual antigen vaccines.Taken together, these data demonstrate the generation of PSMA andPSA-specific T cell responses, as well as PSCA and PSA-specific T cellresponses following dual antigen DNA vaccination, using bothco-expression and co-formulation vaccine strategies.

FIG. 22 shows the results of a representative study that evaluates theimmunogenicity of the dual antigen vaccines by anti-PSMA antibodytiters. Briefly, 5 mice per group were primed on day 0 and boosted withPMED on days 14, 28 and 49. On day 56, serum anti-PSMA antibody titerswere assessed by ELISA. All animals vaccinated with PSMA generatedsignificant anti-PSMA antibody titers. There was no significantdifference in the antibody titers between mice vaccinated withPSA-F2A-PSMA, co-formulated PSMA and PSA, and PSMA alone (one-way ANOVA,p-value >0.05). Taken together, these data demonstrate the generation ofanti-PSMA-specific antibodies following dual antigen DNA vaccinationwith PSMA and PSA, using both co-expression and co-formulation vaccinestrategies.

FIG. 23 shows the results of a representative study that evaluates theimmunogenicity of the dual antigen vaccines by anti-PSCA antibodytiters. Briefly, 5 mice per group were primed on day 0 and boosted withPMED on days 14, 28 and 49. On day 56, serum anti-PSCA antibody titerswere assessed by ELISA. All animals vaccinated with PSCA generatedsignificant anti-PSCA antibody titers. There was no significantdifference in the antibody titers between mice vaccinated withPSCA-IRES-PSA, co-formulated PSCA and PSA, and PSCA alone (one-wayANOVA, p-value >0.05), although the antibody titers generated followingPSCA-IRES-PSA vaccination trended lower than the other groups vaccinatedwith PSCA. These results indicate that co-expression or co-formulationof PSCA and PSA elicits anti-PSCA antibodies.

FIG. 24 shows the results of a representative study that evaluates theimmunogenicity of the dual antigen vaccines by anti-PSMA antibodycell-surface binding. Briefly, 5 mice per group were primed on day 0 andboosted with PMED on days 14, 28 and 49. On day 56, recognition ofcell-surface native PSMA was assessed by serum antibody binding to LNCaPand PC3 cells. The PC3 cells served as a background control for theassay. There were no significant differences in the anti-PSMA antibodybinding to LNCaP cells between the PSMA-vaccinated groups (one-wayANOVA, p-value >0.05). The fold change over secondary antibody alone forthe J591-A mAb was 45.3 (data not shown). Overall, these datademonstrate the generation of anti-PSMA-specific antibodies thatrecognized native PSMA following dual antigen vaccination, using bothco-expression and co-formulation vaccine strategies.

FIG. 25 shows the results of a representative study that evaluates theimmunogenicity of the dual antigen vaccines by anti-PSCA antibodycell-surface binding. Briefly, 5 mice per group were primed on day 0 andboosted with PMED on days 14, 28 and 49. On day 56, recognition ofcell-surface native PSCA was assessed by serum antibody binding toAd-PSCA transduced and untransduced MIA-PaCa-2 cells. The untransduced,parental cells served as a background control for the assay. All groupsof mice vaccinated with PSCA demonstrated very low anti-PSCA antibodybinding to Ad-PSCA transduced MIA-PaCa-2 cells. PSCA-IRES-PSAvaccination resulted in significantly decreased binding to Ad-PSCAtransduced MIA-PaCa-2 cells compared to mice vaccinated with PSCA alone(* indicates p<0.05 by one-way ANOVA). Taken together, these datademonstrate that co-expression or co-formulation of PSCA and PSA resultsin very low recognition of native PSCA by anti-PSCA-specific antibodies.

Example 7 Immunogenicity of the Human Psma Modified Antigen

Study Design.

The immune responses induced by DNA vaccination using a constructencoding an immunogenic PSMA polypeptide (the “human PSMA modifiedantigen” or “hPSMA modified”) consisting 15-750 amino acids (aa) of thenative human PSMA protein of SEQ ID NO: 1 were compared with thoseinduced by the native human full-length PSMA protein (hPSMA fulllength). Groups of female C57BL/6 mice or female Pasteur (HLA-A2/DR1)transgenic mice were primed on day 0 and boosted on days 14, 28 by PMEDadministration with a 2 μg dose of a DNA vaccine encoding either hPSMAfull-length or hPSMA modified protein. Mice were bled and sacrificed onday 35 (7 days after the third vaccination) and T cell immune responsesagainst the hPSMA full-length protein were determined in splenocytes byIFN-γ ELISPOT assay. For C57BL/6 mice, single cell suspensions of 5×10⁵splenocytes from individual animals were plated per well with 10 μgpurified hPSMA protein, 5×10⁴ TRAMP-C2 cells alone, or TRAMP-C2 cellsexpressing hPSMA or a PSMA-PSA-PSCA fusion protein. For Pasteur(HLA-A2/DR1) transgenic mice, single cell suspensions of 5×10⁵splenocytes from individual animals were plated per well with 5×10⁴ K562cells expressing human HLA-A2 that had been pulsed with knownHLA-A2-restricted CD8⁺ T cell epitopes derived from the human PSMAprotein sequence (Table 23). Responses in Pasteur mice were alsodetermined using 10 μg/ml purified PSMA protein or 5×10⁴ SK-Mel5 cellsthat had been transduced with Adenoviral vectors expressing a controlprotein (Ad-eGFP) or the full-length human PSMA protein (Ad-hPSMA). Eachcondition was performed in triplicate. The plates were incubated for 20h at 37° C. and 5% CO₂, washed and developed after incubation as per themanufacturer's instruction. The number of IFN-γ SFC was counted by a CTLreader. The results are presented in FIGS. 19 and 20, which show theaverage number of PSMA-specific SFC/million splenocytes+/−the standarddeviation per group.

ELISA Assay.

Antibody responses induced by the modified and full-length PSMA vaccineswere measured in serum from each animal collected on day 35. Serum fromwas subjected to ELISA to determine the anti-PSMA antibody titers in theserum was determined using the ELISA assay as described in Example 5.The results are presented in FIG. 26, which shows the averagetiters+/−the standard deviation of the number of mice per group.

FACS Assay.

Serum was also subjected to a FACS assay to measure antibody binding toeither human PSMA expressed on the cell surface of appropriate celllines, thus determining whether antibodies generated by the modified andfull-length PSMA vaccines were capable of recognizing native PSMAconformation. The FACS assay was conducted according to the procedure asdescribed in Example 5. The results are presented in FIG. 29, which showthe average fold change in MFI of the mouse serum over the secondaryanti-mouse antibody alone+/−the standard deviation of the number of miceper group.

TABLE 23 HLA-A2 restricted peptide epitopes tested in the assaysconducted for the Pasteur (HLA-A2/DR1) transgenic mice. Peptides weretested individually at a concentration of 10 μg/ml. The amino acidposition of the N and C-terminal end of each peptide is indicated.Prostate antigen Peptides Purpose hHer2 106-114 HLA-A2-restrictedcontrol peptide derived from the human Her2 protein PSMA 168-177HLA-A2-restricted PSMA test peptide PSMA 663-671 HLA-A2-restricted PSMAtest peptide PSMA 275-289 HLA-A2-restricted PSMA test peptide

Results.

FIG. 26 shows the results of a representative study to evaluate the Tcell immune response elicited by the human PSMA modified (aa 15-750)versus full-length human PSMA (aa 1-750) in C57BL/6 mice determined byIFN-γ ELISPOT assay. Five (5) mice per group were primed on day 0 andboosted PMED with DNA vaccines expressing hPSMA modified or hPSMAfull-length proteins on days 14 and 28. On day 35, the response elicitedagainst the hPSMA full-length protein were compared by determining Tcell responses to TRAMP C2-PSMA or purified human PSMA ECD protein(referred to Purified hPSMA protein in FIG. 26) by IFNγ ELISPOT assay.TRAMP C2 cells served as a background control for the assay. Themagnitude of the IFN-γ T cell responses elicited to TRAMP C2-PSMA orpurified hPSMA protein were not significantly different (two-way ANOVA,p-value >0.05) between groups. These results indicate that the DNAvaccines expressing hPSMA modified and hPSMA full-length proteins elicitequivalent T cell immune responses in C57BL/6 mice.

FIGS. 27A and 27B show the results of a representative study to evaluatethe T cell immune response of human PSMA modified antigen (aa 15-750)versus full-length human PSMA antigen (aa 1-750) in Pasteur (HLA-A2/DR1)transgenic mice by IFN-γ ELISPOT assay. Ten (10) mice per group wereprimed on day 0 and boosted PMED with DNA vaccines encoding hPSMAmodified or hPSMA full-length protein on days 14 and 28. On day 35, theT cell response elicited against the hPSMA full-length protein wasdetermined by IFN-ELISPOT assay using (A) PSMA derived HLA-A2-restrictedpeptides representing known CD8⁺ epitopes and (B) SK-Mel5 cellstransduced with Ad-hPSMA or purified hPSMA full-length protein. ThehHER2 106 peptide and SK-Mel5 Ad-eGFP served as negative controls in theassays. The hPSMA modified vaccine elicited the highest magnitude ofIFN-γ T cell immune responses to the HLA-A2-restricted CD8⁺ T cellepitopes, although the difference between groups was not significant(two-way ANOVA, p-value >0.05). Similarly, the hPSMA modified vaccineelicited the highest magnitude of immune response against the SK-Mel5cells transduced with Ad-hPSMA and significantly (two-way ANOVA,p-value >0.05) higher frequencies of IFN-γ SFC to the purified hPSMAprotein. These results demonstrate that the DNA vaccine expressing thehPSMA modified protein is more potent in inducing T cell responses tothe hPSMA protein than the hPSMA full-length protein in Pasteur(HLA-A2/DR1) transgenic mice.

FIG. 28 shows the results of a representative study that evaluates theimmunogenicity of the human modified and full-length PSMA vaccines byanti-PSMA antibody titers. Briefly, mice were primed on day 0 andboosted with PMED on days 14 and 28. Nine Pasteur mice were vaccinatedwith modified PSMA, 10 Pasteur mice were vaccinated with full-lengthPSMA, and 5 C57BL/6 mice per group were vaccinated with either modifiedor full-length PSMA. On day 35, serum anti-PSMA antibody titers wereassessed by ELISA. As expected, C57BL/6 mice generated significantlygreater anti-PSMA antibody titers compared to Pasteur mice (one-wayANOVA). Comparing antibody titers between the same strains of mice,there was no significant difference in the antibody titers between micevaccinated with modified and full-length PSMA (one-way ANOVA,p-value >0.05). Overall, these results demonstrate that vaccination withthe full-length version of human PSMA generates an equivalent anti-PSMAantibody titer compared to the human modified PSMA vaccine.

FIG. 29 shows the results of a representative study that evaluates theimmunogenicity of the human modified and full-length PSMA vaccines byanti-PSMA antibody cell-surface binding. Briefly, 5 C57BL/6 mice pergroup were primed on day 0 and boosted with PMED on days 14 and 28. Onday 35, recognition of cell-surface native PSMA was assessed by serumantibody binding to LNCaP and PC3 cells. The PC3 cells served as abackground control for the assay. There were no significant differencesin the anti-PSMA antibody binding to LNCaP cells between mice vaccinatedwith modified or full-length PSMA (one-way ANOVA, p-value >0.05). Thefold change over secondary antibody alone for the J591-A mAb was 14.3(data not shown). Overall, these data demonstrate that it is feasible togenerate anti-PSMA-specific antibodies that recognized native PSMAfollowing either modified or full-length PSMA vaccination.

Example 8 Effect of Anti-CTLA-4 Antibody on Vaccine-Induced ImmuneResponse

The effect of local administration of anti-CTLA-4 monoclonal antibody(CP-675, 206) on the immune responses induced by a human PSMA nucleicacid molecule provided by the invention was investigated in a monkeystudy, in which the immune response was assessed by measuring PSMAspecific T cell responses using an IFNγ ELISPOT assay.

Animal Treatment and Sample Collection.

Three groups of male Indian rhesus macaques, five to six (#1 to 5 or 6)per each test group, were immunized with a nucleic acid (SEQ ID NO: 10)that encodes a human PSMA modified antigen (SEQ ID NO: 9) delivered byadenovirus (1e11 V.P. injected intramuscularly) followed by 2 DNAimmunizations (8 actuations/immunization, 4 actuations per each rightand left side of the lower abdomen) by PMED with 6 and 9 week intervalsrespectively. Animals in Groups 2 and 3 additionally received bilateralintradermal injections of 3 mg of CpG (PF-03512676) subsequently afterthe PMED immunization in proximity to each inguinal draining lymph node.Group 2 also received intravenous injections of anti-CTLA-4 monoclonalantibody (CP-675, 206) at 10 mg/kg and group 3 received intradermalinjections of anti-CTLA-4 monoclonal antibody (CP-675, 206) at 5 mg/kgin proximity to each left and right inguinal vaccine draining lymph nodeat the time of the second PMED immunization.

Peripheral blood samples were collected from each animal sixteen daysafter the last PMED immunization. Peripheral blood mononuclear cells(PBMCs) were isolated from the samples and were subjected to an IFNγELISPOT assay to measure the PSMA specific T cell responses. Briefly,4e5 PBMCs from individual animals were plated per well with pools ofPSMA specific peptides each at 2 ug/ml hPSMA ECD protein at 10 ug/ml,rhesus PSMA ECD protein at 10 ug/ml or nonspecific control peptides(human HER2 peptide pool) each at 2 ug/ml in IFNγ ELISPOT plates. Thecomposition of each of the PSMA specific peptide pools is provided inTable 24A. The plates were incubated for 16 hrs at 37° C. and 5% CO2 andwashed and developed after incubation as per manufacturer's instruction.The number of IFNγ spot forming cells (SFC) were counted by CTL reader.Each condition was performed in duplicates. The results are presented inTable 24B, which shows the average number of the PSMA specific SFC fromthe triplicates subtracting the average number of SFC from thenonspecific control peptides normalized to 1e6 PBMCs. A indicates thatthe count is not accurate because the numbers of spots were too numerousto count.

IFNγ ELISPOT Assay Procedure.

A capture antibody specific to IFNγ (BD Bioscience, #51-2525kc) iscoated onto a polyvinylidene fluoride (PVDF) membrane in a microplateovernight at 4° C. The plate is blocked with serum/protein to preventnonspecific binding to the antibody. After blocking, effector cells(such as splenocytes isolated from immunized mice or PBMCs isolated fromrhesus macaques) and targets (such as PSMA peptides from peptidelibrary, target cells pulsed with antigen specific peptides or tumorcells expressing the relevant antigens) are added to the wells andincubated overnight at 37° C. in a 5% CO₂ incubator. Cytokine secretedby effector cells are captured by the coating antibody on the surface ofthe PVDF membrane. After removing the cells and culture media, 100 μl ofa biotinylated polyclonal anti-humanlFNy antibody was added to each ofthe wells for detection. The spots are visualized by addingstreptavidin-horseradish peroxidase and the precipitate substrate,3-amino-9-ethylcarbazole (AEC), to yield a red color spot as permanufacturer's (Mabtech) protocol. Each spot represents a singlecytokine producing T cell.

Results.

Table 24B. shows the results of a representative IFNγ ELISPOT assay thatevaluates and compares the T cell responses induced by the vaccinewithout (group 1) or with anti-CTLA-4 monoclonal antibody (CP-675, 206)given either systemically by intravenous injections (group 2) or locallyby intradermal injections in proximity to the vaccine draining lymphnode (group 3). As shown in Table 1B, PSMA vaccine induced measurableIFNγ T cell responses to multiple PSMA specific peptides and proteins inthe absence of CpG (PF-03512676) and anti-CTLA-4 monoclonal antibody(CP-675, 206). The responses were modestly enhanced by the addition ofCpG (PF-03512676) and systemic delivery of the anti-CTLA-4 antibody(CP-675, 206; group 2). However, a more potent and significantenhancement of the response to multiple PSMA peptides and PSMA proteinwas observed when the anti-CTLA-4 monoclonal antibody (CP-675, 206) wasdelivered locally by intradermal injections in proximity to the vaccinedraining lymph node (group 3).

TABLE 24A PSMA peptide pools: Each peptide pool (i.e., P1, P2, P3, H1,H2, R1, and R2) is composed of 15mer peptides from either human PSMAprotein (hPMSA protein) or rhesus PSMA protein (rPMSA protein) sequencesas indicated below. The amino acid position of the N and C-terminal endof each peptide is indicated. P1 P2 P3 H1 H2 R1 R2 h 1-15 h 249-263 h449-463 h 33-47 h 465-479 r 33-47 r 465-479 h 5-19 h 253-267 h 453-467 h37-51 h 469-483 r 37-51 r 469-483 h 9-23 h 257-271 h 457-471 h 41-55 h473-487 r 41-55 r 473-487 h 13-27 h 261-275 h 485-499 h 45-59 h 477-491r 45-59 r 477-491 h 17-31 h 265-279 h 489-503 h 61-75 h 481-495 r 61-75r 481-495 h 21-35 h 269-283 h 493-507 h 65-79 h 537-551 r 65-79 r537-551 h 25-39 h 273-287 h 497-511 h 69-83 h 541-555 r 69-83 r 541-555h 29-43 h 277-291 h 501-515 h 73-87 h 545-559 r 73-87 r 545-559 h 49-63h 281-295 h 505-519 h 97-111 h 577-591 r 97-111 r 577-591 h 53-67 h285-299 h 509-523 h 101-115 h 581-595 r 101-115 r 581-595 h 57-71 h289-303 h 513-527 h 105-119 h 585-599 r 105-119 r 585-599 h 77-91 h293-307 h 517-531 h 109-123 h 589-603 r 109-123 r 589-603 h 81-95 h297-311 h 521-535 h 137-151 h 601-615 r 137-151 r 601-615 h 85-99 h317-331 h 525-539 h 141-155 h 605-619 r 141-155 r 605-619 h 89-103 h321-335 h 529-543 h 145-159 h 609-623 r 145-159 r 609-623 h 93-107 h325-339 h 533-547 h 149-163 h 613-627 r 149-163 r 613-627 h 113-127 h329-343 h 549-563 h 209-223 h 637-651 r 209-223 r 637-651 h 117-131 h333-347 h 553-567 h 213-227 h 641-655 r 213-227 r 641-655 h 121-135 h353-367 h 557-571 h 217-231 h 645-659 r 217-231 r 645-659 h 125-139 h357-371 h 561-575 h 221-235 h 649-663 r 221-235 r 649-663 h 129-143 h361-375 h 565-579 h 301-315 h 653-667 r 301-315 r 653-667 h 133-147 h365-379 h 569-583 h 305-319 h 657-671 r 305-319 r 657-671 h 153-167 h369-383 h 573-587 h 309-323 h 709-723 r 309-323 r 709-723 h 157-171 h373-387 h 593-607 h 313-327 h 713-727 r 313-327 r 713-727 h 161-175 h377-391 h 597-611 h 337-351 h 717-731 r 337-351 r 717-731 h 165-179 h381-395 h 617-631 h 341-355 h 721-735 r 341-355 r 721-735 h 169-183 h385-399 h 621-635 h 345-359 h 725-739 r 345-359 r 725-739 h 173-187 h389-403 h 625-639 h 349-363 h 729-743 r 349-363 r 729-743 h 177-191 h393-407 h 629-643 h 461-475 h 733-747 r 461-475 r 733-747 h 181-195 h397-411 h 633-647 h 185-199 h 401-415 h 661-675 h 189-203 h 405-419 h665-679 h 193-207 h 409-423 h 669-683 h 197-211 h 413-427 h 673-687 h201-215 h 417-431 h 677-691 h 205-219 h 421-435 h 681-695 h 225-239 h425-439 h 685-699 h 229-243 h 429-443 h 689-703 h 233-247 h 433-447 h693-707 h 237-251 h 437-451 h 697-711 h 241-255 h 441-455 h 701-715 h245-259 h 445-459 h 705-719 h 737-750

TABLE 24B T cell responses induced by the vaccine without (Group 1) orwith anti- CTLA-4 antibody Tremelimumab (CP-675, 206) given systemicallyby intravenous injections (Group 2) or local intradermal injections(Group 3). recall antigen animal hPSMA rPSMA Group ID P1 P2 P3 H1 H2protein R1 R2 protein 1. #1 7.5 62.5 0.0 210.0 172.5 455.0 3.8 1.3 0.0no #2 11.3 48.8 0.0 17.5 146.3 111.3 0.0 3.8 0.0 immune #3 12.5 342.513.8 115.0 517.5 705.0 12.5 40.0 138.8 modulator #4 6.3 23.8 0.0 211.338.8 45.0 5.0 7.5 7.5 #5 0.0 16.3 0.0 0.0 52.5 45.0 0.0 0.0 0.0 #6 6.3442.5 21.3 42.5 238.8 736.3 11.3 8.8 93.8 2. #1 23.8 57.5 1.3 71.3 292.5278.8 6.3 6.3 0.0 with #2 0.0 61.3 0.0 2.5 108.8 78.8 0.0 6.3 3.8 aCTLA4#3 58.8 41.3 7.5 1063.8 82.5 1197.5 22.5 7.5 1.3 (IV) #4 25.0 318.8 27.5147.5 983.8 1046.3 26.3 86.3 2.5 #5 15.0 312.5 5.0 402.5 573.8 707.597.5 25.0 20.0 3. #1 48.8 1236.3{circumflex over ( )} 38.8 405.01236.3{circumflex over ( )} 1236.8{circumflex over ( )} 218.8 490.01120.0 with #2 113.8 946.3 17.5 293.8 1247.5{circumflex over ( )}1247.5{circumflex over ( )} 162.5 86.3 5.0 aCTLA4 #3 16.31248.8{circumflex over ( )} 6.3 465.0 1248.8{circumflex over ( )}1248.8{circumflex over ( )} 187.5 295.0 11.3 (ID) #4 6.3 828.8 6.31006.3 1247.5{circumflex over ( )} 1247.5{circumflex over ( )} 142.530.0 17.5 #5 152.5 566.3 18.8 757.5 1173.8 1242.5{circumflex over ( )}287.5 57.5 110.0

Example 9 Systemic Exposure of CTLA-4 Antibody after Administration inMonkeys

The blood levels of anti-CTLA-4 antibody Tremelimumab (CP675206) wereinvestigated in Indian Rhesus macaques after the antibody wasadministered by intradermal or intravenous injections.

Animal Treatment and Sample Collection.

Three animals per treatment group were injected with the anti-CTLA-4antibody Tremelimumab at 10 mg/kg, either with a single intravenousinjection into the saphenous vein or multiple 0.2 ml intradermalbilateral injections in the upper thigh in proximity to the inguinaldraining lymph nodes. Blood samples were collected at 0, 1, 2, 4, 8, 12,24, and 48 hrs post injection into 2.0 ml vaccutainer tubes containinglithium heparin as the anticoagulant. Plasma was collected from thesupernatant in the vaccutainer tubes after centrifugation at 1500×g at4° C. for 10 min. The levels of Tremelimumab in the plasma was measuredby a quantitative ELISA assay according to the procedure provided below.

Tremelimumab Quantitative ELISA Assay Procedure.

The 384-well high bind assay plates (VWR-Greiner Bio-One Cat#82051-264)were coated with 25 μl/well of CD-152 (CTLA-4; Ancell ImmunologyResearch Products Cat#501-020) at 1.0 μg/ml in 100 mMcarbonate-bicarbonate coating buffer and incubated overnight at 4° C.Plates were washed ×6 with 1×PBS-Tween (0.01M PBS pH 7.4/0.05% Tween 20)and blocked using 40 μL/well of 5% FBS/1×PBS-Tween and incubated shakingat 600 rpm RT for 1 hour. Standards were prepared by making thefollowing dilutions of Tremelimumab: 200, 67, 22, 7.4, 2.5, 0.82, 0.27,0.09 and 0.03 ng/mL. The samples were diluted to 1:100, 1:1,000 and1:10,000. The diluent consisted of 1% naive cynomolgus macaque sera and5% FBS in 1×PBS-Tween (0.01M PBS pH 7.4/0.05% Tween 20). 25 μL/well ofeach standard, sample and diluent control were transferred in duplicateinto the plate and incubated shaking at 600 rpm RT for 1 hour. Afterwashing ×6 with 1×PBS-Tween, 25 μL/well of secondary antibody (goatanti-human IgG HRP, Southern Biotech Cat#9042-05) at a 1:5,000 dilutionwith 1×PBS-Tween was added and then incubated shaking at 600 rpm roomtemperature for 1 hour. After washing ×6 with 1×PBS-Tween, 25 μL/well ofTMB Peroxidase EIA-Substrate (solution A+B) (Bio-Rad Cat#172-1067) wereadded and the plates were incubated at RT for 4 minutes. Thecolorimetric reaction was stopped by addition of 12.5 μL/well 1NSulfuric acid and the absorbance then read at 450 nm. The amount ofTremelimumab in each sample was quantified using the standard curve with0.27 to 67 ng/mL used as the quantitative range.

Results.

The plasma anti-CTLA-4 levels from a representative study are presentedin FIG. 30. As shown, intradermal injection of the anti-CTLA-4 antibodyTremelimumab displays a slower release kinetics of the antibody in theblood and a lower systemic exposure (AUC₀₋₂₄=4.9×10⁶ ng·hr/ml) profilethan intravenous injections (AUC₀₋₂₄=7.2×10⁶ ng·hr/ml).

Example 10 Effect of Anti-CTLA-4 Antibody on Vaccine-Induced ImmuneResponses in Mice

Study Procedure.

Female BALB/c mice, 6 per group, were primed and boosted with rHer-2expressing DNA by PMED separated by a four week interval. 150 μg of themonoclonal antibody specific to mouse CTLA-4 (clone 9H10, Bioxcell or#BE0131) or isotype control monoclonal antibodies (Bioxcell #BE0091) wasadministered on the days of PMED actuation and 100 μg on the days afterPMED by local intradermal or systemic intraperitoneal injections asindicated in the legends. The polyfunctional (multi-cytokine positive) Tcell immune responses were measured from splenocytes isolated fromindividual mice 7 days after the last PMED immunizations by ICS assay.After a 5 hr stimulation with a vaccine specific epitope peptide (rHer-2specific antigen specific CD8 (p66), CD4 (p169) epitope or irrelevantpeptide HBV (core antigen p87)) at 10 μg/ml, the splenocytes were firststained for CD4, CD3 and CD8 which was followed by permeabilization andstaining for IFNα, TNFα and IL-2 expression that was analyzed by flowcytometry. The total number of antigen specific single, double or triplecytokine positive T cells per total spleen of each animal is calculatedby subtracting the responses to the irrelevant peptide HBV from thevaccine specific responses and normalized by the total number ofsplenocytes isolated per spleen.

Results.

FIGS. 31A and 31B show the results of a representative study thatevaluates the immunomodulatory activity of anti-CTLA-4 monoclonalantibody (clone 9H10) on the quality of the vaccine induced immuneresponses by intracellular cytokine staining assay. Seven days after thelast PMED, significant increases in antigen specific single and doublecytokine positive CD8 T cell responses by the local intradermal deliveryof anti-CTLA-4 and double and triple cytokine positive CD8 T cellresponses by the systemic delivery was observed. Additionally,significant increases in antigen specific single cytokine positive CD4 Tcells by intradermal delivery and double cytokine positive cells bysystemic delivery of anti-CTLA-4 was observed (*indicates P<0.05 byStudent's T-test).

Example 11 Synergistic Effect of Sunitinib in Combination with anAnti-Cancer Vaccine

Study Procedure.

The Anti-tumor efficacy of sunitinib malate in combination with ananti-cancer DNA vaccine was investigated in BALB/neuT transgenic femalemice. Heterozygote BALB/neuT transgenic female mice that express ratHer-2 (rHer-2) tumor associate antigen were implanted subcutaneouslywith 1e6 TUBO cells expressing rHer-2 which are derived from thespontaneous mammary tumors of BALB/neuT mice. After 7 days post tumorcell implantation, the mice were dosed once a day orally with eithervehicle or sunitinib malate at doses as indicated in the legends. Threedays after the initiation of sunitinib malate therapy, the mice wereimmunized with regimens comprised of either (a) control vaccine thatexpresses an antigen that is not expressed in the tumor or the mouse or(b) DNA cancer vaccine construct that expresses a rat Her-2 antigen ofSEQ ID NO: 54 (rHER2) which is expressed in the tumor and the mouse. Thetumor growth rate was analyzed by measuring the long (a) and shortdiameter (b) of the subcutaneous TUBO tumors twice a week andcalculating the volume as a×b²×0.5 mm³. The average and standard errorof the mean of the tumor volumes from 10 mice per each treatment groupare plotted against the days after tumor implantation.

Results.

FIG. 32 shows the results of a representative study that evaluates andcompares the subcutaneous tumor growth rate upon treatment withsunitinib malate as a monotherapy or in combination with the DNA cancervaccine. While the tumors from mice that received the DNA cancer vaccine(rHER2: intramuscular injection of 1e9 V.P. of rHer-2 expressingadenovirus followed by two biweekly actuations of rHer-2 expressing DNAby PMED) continued to grow rapidly, the tumors from mice that receivedsunitinib malate at either 20 mg/kg or 40 mg/kg doses with controlvaccines (control: intramuscular injection of 1e9 V.P. of eGFPexpressing adenovirus followed by two biweekly, actuations of HBV coreantigen expressing DNA by PMED) significantly decreased the tumor growthrate, with 20 mg/kg displaying suboptimal efficacy compared to the 40mg/kg dose. However when the cancer vaccine was co-administered with thesuboptimal dose of sunitinib malate at 20 mg/kg, the tumors grew at amuch slower growth rate than in mice treated with the same dose ofsunitinib malate co-administered with a control vaccine and similar tothat of mice treated with sunitinib malate at a higher dose. Cancervaccine provides additional therapeutic benefit to mice that receivedsuboptimal doses of sunitinib malate.

FIGS. 33A-33D show the individual tumor growth rates of mice from arepresentative study that evaluates and compares the anti-tumor efficacyof sunitinib malate at 20 mg/kg with control (control) or the DNA cancervaccine (rHER2). Briefly, after 7 days post tumor cell implantation, tenmice per treatment group were daily orally dosed with either vehicle or20 mg/kg sunitinib malate (Sutent) for 34 days. Three days after theinitiation of Sutent dosing, a series of immunizations, primed byAdenovirus followed by PMED, were initiated that continued after thediscontinuation of Sutent therapy. Specifically the mice were immunizedwith either control vaccine comprised of an intramuscular injection of1e9 V.P. of eGFP expressing adenovirus subsequently followed by twobiweekly, two 9 days and four weekly actuations of DNA expressing HBVcore and surface antigens by PMED or cancer vaccine comprised of rHer-2expressing adenovirus and DNA instead. The tumors of the animals thatreceived vehicle with the control vaccine became measurable around day 7and continued growing reaching 2000 mm³ after 50 days post tumorimplant. The tumor growth of the animals that received Sutent withcontrol vaccine was significantly impaired until Sutent therapy wasdiscontinued. The tumors displayed a rapid growth rate immediately afterthe discontinuation of Sutent, the majority reaching 2000 mm³ after 50days post tumor implant. The tumor growth rate of animals that onlyreceived the cancer vaccinations was modestly slower than the animalsthat did not receive cancer vaccine or Sutent. The combination of cancervaccine with Sutent not only suppressed the tumor growth during Sutenttherapy (FIG. 33) but also significantly impaired the progression of thetumor in 60% of the animals after discontinuation of Sutent treatment.

FIG. 34 shows the Kaplan-Meier survival curve of the groups of mice fromthe study described in FIGS. 33A-33D FIG. 2B that evaluates theanti-tumor efficacy of Sutent with the control (control) or cancervaccine (rHER2). The mice were sacrificed when the tumor volume reached2000 mm³ according to IAUCUC guidelines. Only mice treated with Sutent(at 20 mg/kg) and cancer vaccine displayed a significantly prolongedsurvival compared to mice either treated with vehicle and controlvaccine, cancer vaccine without Sutent or Sutent without cancer vaccine(*P<0.01 by Log-rank Test).

FIGS. 35A-35D show the percentage of myeloid derived suppressor cells(Gr1+CD11b+) and Treg containing CD25+CD4+ cells in the periphery bloodfrom the groups of mice from the study described in FIGS. 33A-33D.Briefly, PBMCs were stained and analyzed by flow cytometry for theexpression of Gr1, CD11b, CD3, CD4, and CD25 from submandibular bleedsof five mice from each group on d27 (20 days post the initiation ofSutent or vehicle treatment). The mean and standard error of the mean ofeach treatment group is shown. A statistically significant reduction of% myeloid derived suppressor cells was observed in mice that weretreated by Sutent with either control or cancer vaccine compared to micethat did not receive Sutent nor cancer vaccine (vehicle+control).However significantly lower myeloid derived suppressor cells wereobserved in mice treated with the combination of Sutent with cancervaccine (Sutent+rHER2) compared to mice that were treated with cancervaccine without Sutent (vehicle+rHER2) or Sutent without cancer vaccine(Sutent+control). A statistically significant reduction of Tregcontaining CD25+CD4+T cells in the CD4 population was observed by Sutentwith cancer vaccine. These mice had significantly lower % of Tregcontaining CD25+CD4+T cells in the CD4 population than mice that weretreated with cancer vaccine without Sutent or Sutent without cancervaccine in their blood. * indicates P<0.05 by Student's T-test.

FIGS. 36A-36C show the total number of myeloid derived suppressor cells(Gr1+CD11b+), Tregs (CD4+CD25+Foxp3+) and PD-1+CD8 T cells isolated fromtumors of mice. Briefly, the mice were given a single daily oral dose ofeither vehicle or Sutent at 20 mg/kg three days after implantation withTUBO cells for 28 days. The same mice were immunized with either controlvaccine comprised of an intramuscular injection of 1e9 V.P. of eGFPexpressing adenovirus subsequently followed by two biweeklyadministrations of DNA expressing HBV core antigen delivered by PMED orcancer vaccine comprised of rHer-2 expressing adenovirus and DNA. Anintradermal injection of 50 μg of CpG (PF-03512676) was given with thePMED administrations in proximity to the right side inguinal draininglymph node. Seven days after the second PMED and CpG administration,individual tumors were isolated, from 6 mice per treatment group. Thesingle cell suspension prepared from the isolated subcutaneous tumorswere stained by antibodies specific for Gr1, Cd11 b, CD3, CD8, CD25,FoxP3, and PD-1 and analyzed by flow cytometry. The mean and standarderror of the mean of the total number of specific cells as indicated inthe figures per μg of tumor from each treatment group is plotted.(*indicates P<0.05 by Student's T-test) While there was no significantdifference in the frequency of immune suppressive Tregs or MDSC found inthe tumor when mice were given cancer vaccine (A and B) compared to micethat received control vaccine (A and B), a significant reduction wasobserved when the mice were treated with Sutent (A and B) compared tomice that received cancer vaccine only. A reduction of PD-1+CD8 T cellswas also observed in mice that were treated with Sutent (C) compared tomice that received cancer vaccine (C) only. Taken together, these datademonstrate that agents that reduce Tregs, MDSCs or CD8+PD-1+Tcells incombination with the vaccine would be beneficial in reducing tumorburden in tumor bearing animals.

Example 12 Anti-Cancer Efficacy of Vaccine in Combination with Sunitiniband Anti-CTLA-4 Antibody

The anti-tumor efficacy of a cancer vaccine in combination withsunitinib and anti-CTLA-4 monoclonal antibody (clone 9D9) wasinvestigated in subcutaneous TUBO tumor bearing BALB/neuT mice.

Study Procedure.

Briefly, ten mice per each group were daily orally dosed with eithervehicle or sunitinib malate at 20 mg/kg starting at day 10 post tumorimplant until day 64. Vaccination with DNA constructs that either encodeno antigen (control vaccine) or a rat Her-2 antigen of SEQ Id NO: 54(cancer vaccine) as adenovirus vectors initiated on day 13 subsequentlyfollowed by two weekly immunizations, two biweekly immunizations, andseven weekly immunizations of respective antigens (HBV antigens orrHer-2) by DNA. The groups of mice (closed circle and open triangle)that were treated with anti-murine CTLA-4 monoclonal antibody wereintraperitoneally injected with 250 μg of the antibody on day 20, 27,41, 55, 62, 69, 76, 83, 90, and 97 right after the PMED injections.

Results.

FIG. 37 shows the Kaplan-Meier survival curve of the groups of mice froma representative study evaluating the anti-tumor efficacy of sunitiniband anti-murine CTLA-4 monoclonal antibody (clone 9D9) in combinationwith a cancer vaccine. Increased survival time was observed in micetreated with Sutent with control vaccine (open circle), anti-murineCTLA-4 monoclonal antibody (open triangle) or cancer vaccine (closedtriangle). A further increase of survival was observed in mice treatedwith Sutent and cancer vaccine in combination with anti-murine CTLA-4(closed circle). P values were calculated by log-rank test.

Example 13 Systemic Exposure of Sunitinib and Anti-Cancer Efficacy ofAnti-Cancer Vaccine in Combination with Low Dose Sunitinib

Sunitinib Systemic Exposure Study.

The kinetics of blood sunitinib was investigated in BALB/neuT mice withsubcutaneous TUBO tumors. Briefly, 20 mice per each treatment group weregiven Sutent orally, at 20 mg/kg once a day (SID) or at 10 mg/kg twice aday (BID) with 6 hr intervals, 6 days after tumor implantation.Submandibular blood from 2-3 mice was collected into lithium heparintubes at several time points after Sutent dosing as indicated (0, 2, 4,6, 8, 10, 12, and 24 hr). The plasma supernatant was recovered from thetubes after centrifugation at ×1000 g for 15 min. and the sunitiniblevels from the plasma samples were measured by LC/MS/MS. The mean andstandard error of the mean of each group at each time point is plotted.

Results are presented in FIG. 38. The mean and standard error of themean of each group at each time point is plotted. The dotted horizontalline marks the minimum sunitinib blood level, 50 ng/ml, that isnecessary to effectively inhibit tumor growth in monotherapy (Mendel,D., et al.: “In vivo antitumor activity of SU11248, a novel tyrosinekinase inhibitor targeting vascular endothelial growth factor andplatelet-derived growth factor receptors: determination of apharmacokinetic/pharmacodynamic relationship”. Clinical Cancer Research,203, 9:327-337). As shown, the blood suntinib levels in the mice thatreceived either 20 mg/kg SID or 10 mg/kg BID only maintain the targeteffective dose of 50 ng/ml that effectively inhibits tumor growthtransiently within 24 hr. The blood levels in 20 mg/kg SID group peakedabove 50 ng/ml at 2 hrs, dropped to 50 ng/ml at 6 hrs and cleared theblood by 12 hrs post Sutent dosing he levels in the group that received10 mg/kg BID peaked above 50 ng/ml at 2 hrs but rapidly dropped below 50ng/ml by 4 hrs that peaked again 2 hrs after the second dose. The levelsrapidly dropped to 50 ng/ml by 4 hrs and cleared the blood by 18 hrsafter the second dose. Despite the bi-daily dosing regimen, the animalsthat received 10 mg/kg, remained to display lower duration of exposureat target concentration than the 20 mg/kg single daily dosing regimen.

Anti-Tumor Efficacy Study.

Anti-tumor efficacy of long term administration of low dose sunitinib incombination with an anti-cancer vaccine was investigated in BALB/neuTmice with subcutaneous TUBO tumors. Briefly, the mice were givensunitinib malate (Sutent) for 31 days at 20 mg/kg SID or for 104 days at10 mg/kg BID and received either control vaccine or cancer vaccine. Thecontrol vaccine, which delivers no antigen, and the cancer vaccine,which delivers a rat her-2 antigen (rHer-2) of SEQ ID NO: 54, was givenby adenovirus on day 9 subsequently followed by five biweeklyadministrations of the DNA by PMED delivering HBV antigens or rHer-2respectively.

The results are presented in FIG. 39. While the cancer vaccine improvedthe survival of mice given Sutent at 20 m/kg, there was even significantimprovement of the survival of mice given Sutent at 10 mg/kg (*P=0.05 byLog rank test).

Example 14 Effect of CpG or CD40 Agonist on the Immune Responses Inducedby Cancer Vaccine

Immunogenicity Studies in BALB/c Mice

The effect of local administration of immune modulators on the magnitudeand quality of antigen specific immune responses induced by a cancer wasinvestigated in BALB/c mice, in which the immune response was assessedby measuring rHER2 specific T cell responses using the IFNγ ELISPOTassay or intracellular cytokine staining assay. Briefly, 4 to 6 femaleBALB/c mice per group as indicated were immunized with DNA plasmidexpression constructs encoding rHER2 antigen sequences (SEQ ID NO:54) byPMED delivery system. The immune modulators, CpG7909 (PF-03512676) andanti-CD40 monoclonal agonistic antibody, were administered locally byintradermal injections in proximity to the vaccine draining inguinallymph node subsequently after the PMED actuations. Antigen specific Tcell responses were measured by IFNγ ELISPOT or intracellular cytokinestaining assay according to the procedure described below.

Intracellular Cytokine Staining (ICS) Assay

The rHer-2 specific polyfunctional (multi-cytokine positive) T cellimmune responses were measured from splenocytes or PBMCs isolated fromindividual animals by ICS assay. Typically 1e6 splenocytes wereincubated with Brefeldin A at 1 μg/ml and peptide stimulant (rHer-2specific CD8 p66, rHer-2 specific CD4 p169 or irrelevant HBV p87) at 10μg/ml for 5 hr at 37° C. in a 5% CO₂ incubator. After the stimulation,the splenocytes were washed and blocked with Fcγ block (anti-mouseCD16/CD32) for 10 min. at 4° C. followed by a 20 min staining withLive/dead aqua stain, anti-mouse CD3ePE-Cy7, anti-mouse CD8a Pacificblue, and anti-mouse CD45R/B220 PerCP-Cy5.5. The cells were washed,fixed with 4% paraformaldehyde overnight at 4° C., permeabilized with BDfix/perm solution for 30 min at RT and incubated with anti-mouse IFNγAPC, anti-mouse TNFα Alexa488 and anti-mouse IL-2 PE for 30 min at RT.The cells were washed and 20, 000 CD4 or CD8 T cells were acquired foranalysis by flow cytometry. The total number of antigen specific single,double or triple cytokine positive T cells per total spleen of eachanimal is calculated by subtracting the rHer-2 specific responses to theirrelevant peptide HBV from the vaccine specific responses andnormalized to the total number of splenocytes isolated from the spleen.

IFNγ ELISPOT Assay Results

FIG. 40 shows the IFNγ ELISPOT results from groups of mice from arepresentative study evaluating the magnitude of antigen specific T cellresponses induced by the rHER2 vaccine when given with the immunemodulators as indicated. Briefly, each mouse per treatment group (n=4)was immunized with DNA plasmid expression constructs encoding rHER2antigen sequences (SEQ ID NO:54) by PMED immediately followed by either100 ug of control rat IgG monoclonal antibody (Bioxcell #BE0089: controlmAb) or 50 μg CpG7909 or 100 ug of anti-CD40 monoclonal antibody(Bioxcell #BE0016-2: a-CD40 mAb) as indicated. The antigen specificimmune responses were measured by IFNγ ELISPOT assay from 5e5splenocytes mixed with control or rHer-2 specific p66 peptides at 10μg/ml concentration, 7 days after the PMED actuation. The number oftotal IFNγ secreting cells from splenocytes containing 1e5 CD8 T cellswere calculated from the ELISPOT results from individual animals and the% of CD8 T cells in splenocytes and mean and standard mean of error ofeach group are plotted. As shown, both CpG7909 and the anti-CD40monoclonal antibody both significantly enhanced the magnitude of antigenspecific immune responses induced by rHer-2 DNA compared to mice thatreceived control antibodies.

Intracellular Cytokine Staining (ICS) Assay Results.

FIGS. 41A and 41B show the results of a representative study thatevaluates the immunomodulatory activity of CpG 7909 on the quality ofthe vaccine induced immune responses by intracellular cytokine stainingassay. Briefly, each animal was immunized twice with the DNA plasmidexpression constructs encoding rHER2 antigen sequences (SEQ ID NO:54)delivered by PMED with a 4-week interval. The mice in each group (n=5)were given intradermal injections of either PBS (PMED group) or 50 μg ofCpG 7909 (PMED+CpG group) in proximity to the right side vaccinedraining inguinal node immediately following both DNA immunizations byPMED. Seven days after the last immunization by PMED, an ICS assay wasperformed on the splenocytes isolated from each individual mice todetect antigen specific polyfunctional CD8 or CD4 T cells that secreteIFNγ, TNFα and/or IL-2. A significant increase in rHer-2 specificmulti-cytokine positive CD8 and CD4 T cell responses were detected frommice treated with the local delivery of CpG 7909 compared to PBS. Anincrease in the single cytokine positive CD8 population was observed inthe animals that received local delivery of CpG7909 administrationcompared to PBS (*indicates P<0.05 by Student's T-test).

FIGS. 42A and 42B show the results of a representative study thatevaluates the immunomodulatory activity of an agonistic anti-CD40monoclonal antibody on the quality of the vaccine induced immuneresponses by intracellular cytokine staining assay. Briefly, each animalwas immunized twice by DNA plasmid expression constructs encoding rHER2antigen sequences (SEQ ID NO:54) delivered by PMED with a 4 weekinterval. The mice in each group (n=6) were given 100 μg of intradermalinjections of either isotype IgG control (PMED with IgG) or anti-CD40monoclonal antibody (PMED with aCD40) in proximity to the right sidevaccine draining inguinal node, one day after the first immunization wasadministered by PMED. Seven days after the last PMED, an ICS assay wasperformed on the splenocytes isolated from each individual mice todetect rHer-2 specific polyfunctional CD8 or CD4 T cells that secreteIFNγ, TNFα and/or IL-2. A significant increase in the rHer-2 specifictriple-cytokine positive CD8 and CD4 T cell responses were detected frommice treated with the local delivery of anti-CD40 monoclonal antibodycompared to isotype IgG control. There were also significant increasesin rHer-2 specific single and double cytokine positive CD4 T cells byanti-CD40 monoclonal antibody given locally (*indicates P<0.05 byStudent's T-test).

Example 15 Anti-Cancer Efficacy of Cancer Vaccine in Combination withLow Dose Sunitinib

Anti-tumor efficacy of anti-cancer vaccine in combination with low dosesunitinib was investigated in BALB/neuT mice with spontaneous mammarypad tumors.

Animal Treatment.

Briefly, 13-14 weeks old female mice were orally given sunitinib malate(Sutent) at 5 mg/kg for 112 days twice a day. The control vaccine, whichdelivers no antigen, and cancer vaccine which delivers a rat Her-2antigen of SEQ ID NO: 54 (rHer-2), were given by adenovirus injectionson day 3 as a prime followed by 7 biweekly administrations by PMED ofDNA delivering HBV antigens (control vaccine) or rHer-2 (cancer vaccine)respectively. The survival end point was determined when all ten mammarypads became tumor positive or when the volume of any of the mammarytumors reach 2000 mm³. The results are presented in FIG. 43.

Results.

Compared to previously published pharmacokinetic profile of Sutent(Mendel, D., Laird, D., et al.: “In vivo antitumor activity of SU11248,a novel tyrosine kinase inhibitor targeting vascular endothelial growthfactor and platelet-derived growth factor receptors: determination of apharmacokinetic/pharmacodynamic relationship”. Clinical Cancer Research,203, 9:327-337) and previous data (FIG. 38), the C_(Max) of Sutent inmice dosed twice a day at 5 mg/kg is expected to be significantly lowerthan the minimum blood levels necessary to achieve efficient anti-tumorefficacy in mice and man. The data shows a quick and temporaryimprovement in the survival of the mice treated with low dose Sutentmonotherapy. However when given with the cancer vaccine, a morepersistent and significant improvement of survival was observed(P<0.0001 by Log rank test).

Example 16 Enhancement of Vaccine-Induced Immune Responses by LocalAdministration of CpG

The immune enhancement of local administration of CpG (PF-03512676) onthe immune responses induced by a human PSMA nucleic acid provided bythe invention was investigated in a monkey study, in which the immuneresponse was assessed by measuring PSMA specific T cell responses usingan IFNγ ELISPOT assay.

Animal Treatment and Sample Collection.

Six groups of Chinese cynomolgus macaques, six (#1 to 6) per each testgroup, were immunized with a plasmid DNA encoding the human PSMAmodified antigen (amino acids 15-750 of SEQ ID NO:1) delivered byelectroporation. Briefly, all animals received bilateral intramuscularinjections of 5 mg of plasmid DNA followed by electroporation (DNA EP)on day 0. Subsequently right after the electroporation, group 2 receivedbilateral intramuscular injections of 2 mg of CpG mixed with 1 mg Alumin proximity to the DNA injection sites. Group 3 and 4 receivedbilateral intramuscular injections of 2 mg of CpG delivered without alumin proximity to the DNA injection sites either on day 0 or day 3,respectively. Group 5 received 2 mg of bilateral intradermal injectionsof CpG delivered in proximity to the vaccine draining inguinal nodes onday 3. Group 6 received bilateral injections of 200 μg of CpG mixed withthe DNA solution which was co-electroporated into the muscle on day 0.

IFNγ ELISPOT Assay Procedure.

Peripheral blood samples were collected from each animal fifteen daysafter the DNA immunization. Peripheral blood mononuclear cells (PBMCs)were isolated from the blood samples and were subjected to an IFNγELISPOT assay to measure the PSMA specific T cell responses. Briefly,4e5 PBMCs from individual animals were plated per well with pools ofPSMA specific peptides or nonspecific control peptides (human HER2peptide pool) each at 2 ug/ml in IFNγ ELISPOT plates. The composition ofeach of the PSMA specific peptide pool is provided in Table 1A. Theplates were incubated for 16 hrs at 37° C. and 5% CO2 and washed anddeveloped after incubation as per manufacturer's instruction. The numberof IFNγ spot forming cells (SFC) were counted by CTL reader. Eachcondition was performed in duplicates. The result of a representativeexperiment is presented in Table 1B. The reported PSMA specific responseis calculated by subtracting the average number of the SFC to thenonspecific control peptides (human HER2 peptide pool) from the averagenumber of SFC to the PSMA peptide pools and normalized to the SFCobserved with 1e6 PBMCs. ̂ indicates that the count is not accuratebecause the numbers of spots were too numerous to count. ND indicatesnot determined.

Results.

Table 28 shows the result of a representative IFNγ ELISPOT assay thatevaluates and compares the IFNγ T cell responses induced by the vaccinewithout (group 1) or with CpG (PF-03512676) given locally byintramuscular (groups 2, 3, 4, and 5) or intradermal injections (group6). There results in Table 1B is plotted in FIG. 1. As shown in Table 1Band FIG. 1, the PSMA specific IFNγ T cell responses were detected tomultiple PSMA specific peptide pools in the absence of CpG (PF-03512676)in all six animals (group 1). The total response to the PSMA peptidesmeasured were modestly higher in a few animals that additionallyreceived CpG (PF-03512676) either by intramuscular (group 4: 3/6) orintradermal (group 5: 2/6) injections 3 days after DNA electroporation.However, when CpG was delivered subsequently right after electroporationon the same day (groups 2 and 3), there were several animals that failedto produce high responses (group 2: 4/6 and group 3: 3/6) whether mixedor not mixed with Alum. However higher net responses were detected in4/6 animals when a ten-fold lower dose of CpG was co-electroporated withthe DNA solution into the muscle (group 6) with a statistically higherresponse (P<0.05) to peptide pools H1 and R1 compared to animals thatdid not receive CpG (group 1). The data shows that low dose of CpG caneffectively enhance IFNγ T cell responses induced by a DNA vaccine whenco-electroporated into the muscle.

TABLE 28 PSMA specific IFNγ T cell responses induced by the DNA vaccinewithout (Group 1) or with CpG (Groups 2, 3, 4, 5 and 6) is measured byIFNγ ELISPOT assay from PBMCS, 15 days after DNA electroporation RecallAntigen Group Animal ID P1 P2 P3 H1 H2 R1 R2 1 #1 36 31 1 126 183 5 14#2 6 3 13 61 524 6 141 #3 11 4 8 108 1049 3 56 #4 10 0 13 20 151 13 10#5 8 6 11 39 469 14 18 #6 26 5 0 145 356 8 30 2 #1 3 10 0 15 35 0 0 #2 00 8 4 6 13 0 #3 3 0 0 0 10 11 0 #4 6 209 4 111 414 23 9 #5 15 5 30 171104 68 6 #6 0 0 0 9 9 6 8 3 #1 14 19 8 123 1066 10 60 #2 14 16 20 384393 104 8 #3 0 0 15 0 6 0 0 #4 0 0 0 33 21 0 4 #5 4 91 1 875 {circumflexover ( )}1235 233 109 #6 0 0 0 0 3 0 0 4 #1 0 33 15 1025 {circumflexover ( )}1209 280 90 #2 0 313 3 23 656 6 31 #3 61 120 61 428 1190 143 53#4 0 0 8 599 870 34 111 #5 0 1 8 19 226 10 36 #6 111 55 39 231 613 12199 5 #1 21 9 0 355 1131 73 5 #2 0 0 0 118 233 0 0 #3 0 0 0 18 129 0 0 #40 28 78 68 294 58 8 #5 25 0 28 329 1125 134 5 #6 0 0 0 23 39 4 0 6 #1 00 13 650 1096 270 5 #2 34 1 74 124 474 29 15 #3 0 3 14 684 1074 126 64#4 8 9 0 136 321 49 1 #5 13 23 35 ND {circumflex over ( )}1235 333 195#6 0 0 0 421 {circumflex over ( )}1201 138 29

Example 17 Enhancement of Vaccine-Induced Immune Responses by LocalAdministration of Anti-CTLA-4 Antibody

The effect of low dose subcutaneous administration of anti-CTLA-4monoclonal antibody (CP-675, 206) on the immune responses induced by arhesus PSMA nucleic acid was investigated in a monkey study, in whichthe immune response was assessed by measuring PSMA specific T cellresponses using an IFNγ ELISPOT assay. The rhesus PSMA nucleic acid usedin the study has the sequence as set forth in SEQ ID NO: 56) and encodesan immunogenic PSMA polypeptide of SEQ ID NO: 55.

Animal Treatment and Sample Collection.

Five groups of male Indian rhesus macaques, seven (#1 to 7) per eachtest group, were immunized with an adenovirus encoding a rhesus PSMAmodified polypeptide delivered by bilateral intramuscular injections (2×5e10 V.P.). Immediately following the adenovirus injections, group 1received vehicle, and groups 2 to 4 received bilateral subcutaneousinjections of anti-CTLA-4 antibody (CP-675, 206) at doses 2× 25 mg, 2×16.7 mg and 2× 8.4 mg respectively in proximity to the vaccine draininglymph node.

Nine days after the immunization, peripheral blood mononuclear cells(PBMCs) were isolated from each animal and were subjected to an IFNγELISPOT assay to measure the rhesus PSMA specific T cell responses.Briefly, 4e5 PBMCs from individual animals were plated per well withpools of rhesus PSMA specific peptides (P1, P2, P3 or R1+R2 defined intable 24A) or nonspecific control peptides (human HER2 peptide pool)each at 2 ug/ml in IFNγ ELISPOT plates. The plates were incubated for 16hrs at 37° C. and 5% CO2 and washed and developed after incubation asper manufacturer's instruction. The number of IFNγ spot forming cells(SFC) were counted by CTL reader. Each condition was performed induplicates. The average of the duplicates from the background adjustedSFC of the rhesus PSMA specific peptide pools was normalized to theresponse in 1e6 PBMCs. The individual and sum responses to the peptidepools from each individual animal are presented in Table 29.

IFNγ ELISPOT Assay Procedure.

A capture antibody specific to IFNγ BD Bioscience, #51-2525kc) is coatedonto a polyvinylidene fluoride (PVDF) membrane in a microplate overnightat 4° C. The plate is blocked with serum/protein to prevent nonspecificbinding to the antibody. After blocking, effector cells (such assplenocytes isolated from immunized mice or PBMCs isolated from rhesusmacaques) and targets (such as PSMA peptides from peptide library,target cells pulsed with antigen specific peptides or tumor cellsexpressing the relevant antigens) are added to the wells and incubatedovernight at 37° C. in a 5% CO₂ incubator. Cytokine secreted by effectorcells are captured by the coating antibody on the surface of the PVDFmembrane. After removing the cells and culture media, 100 μl of abiotinylated polyclonal anti-human IFNγ antibody was added to each ofthe wells for detection. The spots are visualized by addingstreptavidin-horseradish peroxidase and the precipitate substrate,3-amino-9-ethylcarbazole (AEC), to yield a red color spot as permanufacturer's (Mabtech) protocol. Each spot represents a singlecytokine producing T cell.

Results.

Table 29. shows the results of a representative IFNγ ELISPOT assay thatcompares the T cell responses induced by the vaccine without (group 1)or with (groups 2-4) anti-CTLA-4 monoclonal antibody (CP-675, 206) givenlocally by subcutaneous injections in proximity to the vaccine draininglymph node. The vaccine generated an immune response (group 1) that wassignificantly enhanced by the local administration of the anti-CTLA-4antibody (CP-675, 206) at a dose of 50 mg (group 2, P=0.001 by Student'sT-test using underestimated values). The response was also significantlyenhanced by low doses of anti-CTLA-4 antibody at 33.4 mg (group 3:P=0.004 by Student T-test using underestimated values) and 16.7 mg(group 4: P=0.05 by Student T-test) respectively. The data suggests thatlow doses of anti-CTLA-4 delivered by subcutaneous injection cansignificantly enhance the vaccine induced immune responses.

TABLE 29 IFNγ T cell responses induced by the vaccine without (Group 1)or with subcutaneous injections of anti-CTLA-4 antibody (CP-675, 206).aCTLA4 peptide pool Group dose, mg animal ID P1 P2 P3 R1 + R2 Sum 1 NA 121 0 0 108 129 2 59 480 28 353 920 3 133 29 359 305 826 4 0 28 1 35 64 541 6 30 99 176 6 1 0 849 169 1019 7 0 0 0 23 23 2 50.0 1 {circumflexover ( )}1105 704 {circumflex over ( )}1116 {circumflex over ( )}1116{circumflex over ( )}4041 2 371 26 661 779 1837 3 393 559 216 198 1366 4{circumflex over ( )}1100 {circumflex over ( )}1100 406 1078 {circumflexover ( )}3684 5 778 325 554 419 2076 6 {circumflex over ( )}1079{circumflex over ( )}1079 844 {circumflex over ( )}1079 {circumflex over( )}4081 7 423 103 535 398 1459 3 33.4 1 {circumflex over ( )}425{circumflex over ( )}425 {circumflex over ( )}425 {circumflex over( )}425 {circumflex over ( )}1700 2 {circumflex over ( )}580 {circumflexover ( )}580 {circumflex over ( )}580 {circumflex over ( )}580{circumflex over ( )}2320 3 TNTC TNTC TNTC TNTC TNTC 4 321 778 370 4091878 5 331 466 311 446 1554 6 545 121 {circumflex over ( )}631{circumflex over ( )}1194 {circumflex over ( )}2491 7 446 299{circumflex over ( )}1078 {circumflex over ( )}1060 {circumflex over( )}2883 4 16.7 1 {circumflex over ( )}964 296 {circumflex over ( )}964{circumflex over ( )}964 {circumflex over ( )}3188 2 76 76 76 76 304 3{circumflex over ( )}984 {circumflex over ( )}984 {circumflex over( )}984 {circumflex over ( )}984 {circumflex over ( )}3936 4 260 489 648{circumflex over ( )}1109 {circumflex over ( )}2506 5 119 45 28 140 3326 55 76 43 198 372 7 146 726 141 400 1413 {circumflex over ( )}indicates that the count is underestimated due to the high spot numbers.TNTC means too numerous to count.

Example 18 Immunomodulation of Myeloid Derived Suppressor Cells by LowDose Sunitinib

The following example is provided to illustrate the immunomodulatoryeffects of low dose sunitinib on Myeloid Derived Suppressor Cells (MDSC)in vivo, in a non-tumor mouse model.

Study Procedures.

To generate MDSC enriched splenocytes, TUBO cells (1×10⁶) were implantedinto the flanks of 5 BALB/neuT mice, and left for approx. 20-30 daysuntil tumor volume reached between 1000-1500 mm³. Mice were thensacrificed, spleens removed and the MDSC enriched splenocytes recovered.Splenocytes were labeled for 10 minutes with 5 μM CFSE, washed with PBSand counted. Labeled cells were subsequently resuspended at 5×10⁷splenocytes/ml in PBS solution and adoptively transferred via an i.v.tail vein injection into naïve BALB/c recipient mice. Three days priorto adoptive transfer, the recipient mice began bi-daily dosing withvehicle or sunitinib malate (Sutent) at 5 mg/kg, 10 mg/kg and 20 mg/kg.Following adoptive transfer, recipient mice continued to receivebi-daily dosing of Vehicle or sunitinib for two further days, afterwhich point the mice were sacrificed, spleens removed, splenocytesrecovered and processed for phenotypic analysis.

Splenocytes were counted and resuspended at 5×10⁶ cells/ml in FACSstaining buffer (PBS, 0.2% (w/v) bovine serum albumin, and 0.02% (w/v)Sodium Azide). For flow cytometry staining of splenocytes, 2.5×10⁶ cellswere first incubated with anti-bodies to CD16/CD32, 10 minutes at 4° C.,to block Fc receptors and minimize non-specific binding. Splenocyteswere then stained for 20 minutes at 4° C. with appropriate fluorophoreconjugated antibodies (Biolegend) to murine cell surface markers. For Tcells (anti-CD3 (Pacific Blue), clone 17A2) and for MDSC (anti-GR-1(APC), clone RB6-8C5 and anti-CD11b (PerCp Cy5.5), clone M1/70). Alive/dead stain was also included. Following antibody incubation,stained splenocytes were washed with 2 mls of FACS buffer, pelleted bycentrifugation and resuspended in 0.2 ml of FACS buffer prior to dataacquisition on a BD CANTO II flow cytometer. To monitor the effect ofSunitinib or Vehicle on the adoptively transferred MDSC survival, wecalculated the percentage of CFSE+,CD3−,GR1+,CD11 b+ in the live,singlet gate. We then determined the number of adoptively transferredMDSC per spleen by calculating what actual cell number the percentagerepresented of total splenocytes count. Data was analyzed by FloJo andGraph pad software.

Results.

The data presented in Table 31 represents the mean number of adoptivelytransferred CSFE+,CD3−,GR1+,CD11b+ cells recovered per spleen(n=7/group), 2 days post adoptive transfer, from mice bi-daily dosedwith either Vehicle or 5 mg/kg, 10 mg/kg and 20 mg/kg Sunitinib. Thedata demonstrates that Sunitinib, dosed bi-daily, in vivo, has animmunomodulatory effect on MDSCs, even when dosed as low as 5 mg/kg,resulting in a statistically significant reduction in the numbersrecovered when compared to the vehicle treated control group.

TABLE 31 Mean number of CFSE+, CD3−, GR1+, CD11b+ MDSCs recovered fromthe spleen, 7 mice per group, and the corresponding standard error.Statistical significance was determined by one-way ANOVA using theDunnett's multiple comparison test, comparing the Sunitinib dosed groupsagainst the 0 mg/kg (vehicle) group. Sunitinib Dose (mg/kg) 0 (Vehicle)5 10 20 MDSC #/spleen 17470 +/− 2017 10980 +/− 1082 4207 +/−338 4440 +/−440 Mean +/− SEM Statistical NA Yes Yes Yes significance, p < 0.5 [[?]]

Example 19 Immunogenicity of Triple Antigen Adenovirus and DNAConstructs

The following example is provided to illustrate the capability of tripleantigen vaccine constructs (either in the form of adenovirus vector orDNA plasmid) expressing three antigens PSMA, PSCA and PSA provided bythe invention to elicit specific T cell responses to all three encodedantigens in nonhuman primates.

In Vivo Study Procedures.

The T cell immunogenicity of five adenovirus vectors each expressingthree antigens (PSMA, PSCA and PSA; Ad-733, Ad-734, Ad-735, Ad-796 andAd-809) provided by the invention were compared to the mix of threeadenovirus vectors each only expressing a single antigen (PSMA, PSA orPSCA), 9 days post prime. The response to single adenovirus expressing asingle antigen (groups 1-3) was evaluated to demonstrate thespecificity. Briefly, Indian rhesus macaques (n=6 for groups 1 and 3,n=7 for group 2 and n=8 for groups 4-9) were intramuscularly injectedwith a total of 1e11 V.P. followed by intradermal injections ofanti-CTLA-4 at 10 mg/kg on the same day. Nine days after the injections,peripheral blood mononuclear cells (PBMCs) were isolated from eachanimal and were subjected to an IFNγ ELISPOT assay to measure the PSMA,PSA and PSCA specific T cell responses.

Thirteen weeks after the adenovirus and anti-CTLA-4 injections when theT cell responses have contracted, the monkeys received DNA (Group 1:PSMA, plasmid 5166; Group 2: PSA, plasmid 5297; Group 3: PSCA, plasmid5259; Group 4: mix of PSMA, PSA and PSCA, plasmids 5166, 5259 and 5297;Group 4: plasmid 457; Group 6: plasmid 458; Group 7: plasmid 459; Group8: plasmid 796 and Group 9: plasmid 809) boost vaccinations delivered byelectroporation. In summary, each animal received a total 5 mg ofplasmid DNA provided by the invention which delivers the same expressioncassette encoded in the adenovirus used in the prime. Nine days afterthe boost vaccination, peripheral blood mononuclear cells (PBMCs) wereisolated from each animal and were subjected to an IFNγ ELISPOT assay.

IFNγ ELISPOT Assay.

Briefly, 4e5 PBMCs from individual animals were plated per well withPSMA specific peptide pools P1, P2, P3 or H1 and H2 (Table 24A), PSAspecific pool 1 or 2 (Table 25), PSCA specific pool (Table 26) ornonspecific control peptides (human HER2 peptide pool) each at 2 ug/mlin IFNγ ELISPOT plates. The plates were incubated for 16 hrs at 37° C.and 5% CO2 and washed and developed after incubation as permanufacturer's instruction. The number of IFNγ spot forming cells (SFC)were counted by CTL reader. Each condition was performed in duplicates.The average of the duplicates from the background adjusted SFC of theantigen specific peptide pools was normalized to the response in 1e6PBMCs. The antigen specific responses in the tables present the sum ofthe responses to the corresponding antigen specific peptides or peptidepools.

Results:

Table 27 represents a study that evaluates the T cell immunogenicity offive different adenoviruses each expressing all three antigens incomparison to the mixture of three adenoviruses each expressing a singleantigen in Indian rhesus macaques by IFNγ ELISPOT. The majority ofanimals that only received Ad-PSMA (group 1) injections induced specificresponses to PSMA but not to PSA or PSCA (Student's T-test, P<0.03. Oneanimal (#4) that induced responses to PSCA preferentially was removedfrom the statistical analysis). The animals that only receivedinjections of Ad-PSA (group 2) induced specific responses to PSA but notto PSMA or PSCA (Student's T-test, P<0.02). The animals that onlyreceived injections of Ad-PSCA (group 3) induced specific responses toPSCA but not to PSMA or PSA (Student's T-test, P<0.03). All fivetriple-antigen expressing adenovirus vectors (groups 5-9) induced IFNγ Tcell responses to all three antigens which the magnitude varied byanimal. The magnitude of the responses to PSCA induced by the tripleantigen expressing adenoviruses were similar to the mix of individualvectors (group 4). However the magnitude of responses to PSMA induced byAd-809 (group 9) and responses to PSA induced by Ad-796 (group 8) wereeach significantly superior to the mix (Student's T-test, P=0.04 andP=0.02) respectively. These results indicate that vaccinating with anadenovirus expressing triple antigens can elicit equivalent or superiorT cell immune responses to vaccinating with the mix of individualadenoviruses in nonhuman primates.

Table 28 shows the IFNγ ELISPOT results represents a study thatevaluates the immunogenicity of the five different triple antigenexpression cassettes provided in the invention delivered by anadenovirus prime in combination with anti-CTLA-4 followed by anelectroporation boost of the corresponding plasmid DNA. The immuneresponses are compared to the mix of three constructs expressing asingle antigen delivered similarly by an adenovirus prime withanti-CTLA-4 and DNA electroporation boost immunizations.

All of the animals that only received Ad-PSMA with anti-CTLA-4 followedby plasmid-PSMA (group 1) immunizations induced specific responses toPSMA but not to PSA or PSCA. Similarly all of the animals that onlyreceived Ad-PSA with anti-CTLA-4 followed by plasmid-PSA immunizations(group 2) induced specific responses to PSA but not to PSMA or PSCA andfinally all of the animals that only received Ad-PSCA with anti-CTLA-4followed by plasmid-PSCA (group 3) immunizations induced specificresponses to PSCA but not to PSMA or PSA (Student's T-test, P<0.01).

All animals that have been immunized with either the triple-antigenexpressing vectors (groups 5-9) or the mix (group 4) induced IFNγ T cellresponses to all three antigens. The frequency of PSCA or PSA specificIFNγ T cells detected were similar in all of these groups (groups 4-9)respectively. However construct groups 7 and 9 that received tripleantigen expression vector vaccinations produced significantly higherfrequency of responses to PSMA than the mix of three single antigenexpressing constructs (group 4). These results indicate that adenovirusand DNA vaccines expressing triple antigens in one cassette can elicitequivalent or superior IFNγ T cell responses to the mix of adenovirusesand DNAs expressing the single antigens in nonhuman primates.

TABLE 25 PSA peptide pools: The amino acid position and sequence offifteen amino acid peptides overlapping by thirteen amino acids from PSApeptide library is shown. PSA peptide pool 1 PSA peptide pool 2 aminoPSA peptide amino PSA peptide acid no. sequence acid no. sequence  5-19VVFLTLSVTWIGAAP 129-143 PAELTDAVKVMDLPT  9-23 TLSVTWIGAAPLILS 131-145ELTDAVKVMDLPTQE 11-25 SVTWIGAAPLILSRI 133-147 TDAVKVMDLPTQEPA 13-27TWIGAAPLILSRIVG 135-149 AVKVMDLPTQEPALG 15-29 IGAAPLILSRIVGGW 137-151KVMDLPTQEPALGTT 17-31 AAPLILSRIVGGWEC 139-153 MDLPTQEPALGTTCY 19-33PLILSRIVGGWECEK 141-155 LPTQEPALGTTCYAS 21-35 ILSRIVGGWECEKHS 143-157TQEPALGTTCYASGW 23-37 SRIVGGWECEKHSQP 145-159 EPALGTTCYASGWGS 25-39IVGGWECEKHSQPWQ 147-161 ALGTTCYASGWGSIE 27-41 GGWECEKHSQPWQVL 149-163GTTCYASGWGSIEPE 29-43 WECEKHSQPWQVLVA 151-165 TCYASGWGSIEPEEF 31-45CEKHSQPWQVLVASR 153-167 YASGWGSIEPEEFLT 33-47 KHSQPWQVLVASRGR 155-169SGWGSIEPEEFLTPK 35-49 SQPWQVLVASRGRAV 157-171 WGSIEPEEFLTPKKL 37-51PWQVLVASRGRAVCG 159-173 SIEPEEFLTPKKLQC 39-53 QVLVASRGRAVCGGV 161-175EPEEFLTPKKLQCVD 41-55 LVASRGRAVCGGVLV 163-177 EEFLTPKKLQCVDLH 43-57ASRGRAVCGGVLVHP 165-179 FLTPKKLQCVDLHVI 45-59 RGRAVCGGVLVHPQW 167-181TPKKLQCVDLHVISN 47-61 RAVCGGVLVHPQWVL 169-183 KKLQCVDLHVISNDV 49-63VCGGVLVHPQWVLTA 171-185 LQCVDLHVISNDVCA 51-65 GGVLVHPQWVLTAAH 173-187CVDLHVISNDVCAQV 53-67 VLVHPQWVLTAAHCI 175-189 DLHVISNDVCAQVHP 55-69VHPQWVLTAAHCIRN 177-191 HVISNDVCAQVHPQK 57-71 PQWVLTAAHCIRNKS 179-193ISNDVCAQVHPQKVT 59-73 WVLTAAHCIRNKSVI 181-195 NDVCAQVHPQKVTKF 61-75LTAAHCIRNKSVILL 183-197 VCAQVHPQKVTKFML 63-77 AAHCIRNKSVILLGR 185-199AQVHPQKVTKFMLCA 65-79 HCIRNKSVILLGRHS 187-201 VHPQKVTKFMLCAGR 67-81IRNKSVILLGRHSLF 189-203 PQKVTKFMLCAGRWT 69-83 NKSVILLGRHSLFHP 191-205KVTKFMLCAGRWTGG 71-85 SVILLGRHSLFHPED 193-207 TKFMLCAGRWTGGKS 73-87ILLGRHSLFHPEDTG 195-209 FMLCAGRWTGGKSTC 75-89 LGRHSLFHPEDTGQV 197-211LCAGRWTGGKSTCSG 77-91 RHSLFHPEDTGQVFQ 199-213 AGRWTGGKSTCSGDS 79-93SLFHPEDTGQVFQVS 201-215 RWTGGKSTCSGDSGG 81-95 FHPEDTGQVFQVSHS 203-217TGGKSTCSGDSGGPL 83-97 PEDTGQVFQVSHSFP 205-219 GKSTCSGDSGGPLVC 85-99DTGQVFQVSHSFPHP 207-221 STCSGDSGGPLVCNG  87-101 GQVFQVSHSFPHPLY 209-223CSGDSGGPLVCNGVL  89-103 VFQVSHSFPHPLYDM 211-225 GDSGGPLVCNGVLQG  91-105QVSHSFPHPLYDMSL 213-227 SGGPLVCNGVLQGIT  93-107 SHSFPHPLYDMSLLK 215-229GPLVCNGVLQGITSW  95-109 SFPHPLYDMSLLKNR 217-231 LVCNGVLQGITSWGS  97-111PHPLYDMSLLKNRFL 219-233 CNGVLQGITSWGSEP  99-113 PLYDMSLLKNRFLRP 221-235GVLQGITSWGSEPCA 101-115 YDMSLLKNRFLRPGD 223-237 LQGITSWGSEPCALP 103-117MSLLKNRFLRPGDDS 225-239 GITSWGSEPCALPER 105-119 LLKNRFLRPGDDSSH 227-241TSWGSEPCALPERPS 107-121 KNRFLRPGDDSSHDL 229-243 WGSEPCALPERPSLY 109-123RFLRPGDDSSHDLML 231-245 SEPCALPERPSLYTK 111-125 LRPGDDSSHDLMLLR 233-247PCALPERPSLYTKVV 113-127 PGDDSSHDLMLLRLS 235-249 ALPERPSLYTKVVHY 115-129DDSSHDLMLLRLSEP 237-251 PERPSLYTKVVHYRK 117-131 SSHDLMLLRLSEPAE 239-253RPSLYTKVVHYRKWI 119-133 HDLMLLRLSEPAELT 241-255 SLYTKVVHYRKWIKD 121-135LMLLRLSEPAELTDA 243-257 YTKVVHYRKWIKDTI 123-137 LLRLSEPAELTDAVK 245-259KVVHYRKWIKDTIVA 125-139 RLSEPAELTDAVKVM 247-261 VHYRKWIKDTIVANP 127-141SEPAELTDAVKVMDL 249-261 YRKWIKDTIVANP 251-261 KWIKDTIVANP

TABLE 26 PSCA peptide pool: The amino acid position and sequence offifteen amino acid peptides overlapping by thirteen amino acids fromPSCA peptide library is shown. amino acid no. PSCA peptide sequence 1-15 MKAVLLALLMAGLAL  3-17 AVLLALLMAGLALQP  5-19 LLALLMAGLALQPGT  7-21ALLMAGLALQPGTAL  9-23 LMAGLALQPGTALLC 11-25 AGLALQPGTALLCYS 13-27LALQPGTALLCYSCK 15-29 LQPGTALLCYSCKAQ 17-31 PGTALLCYSCKAQVS 19-33TALLCYSCKAQVSNE 21-35 LLCYSCKAQVSNEDC 23-37 CYSCKAQVSNEDCLQ 25-39SCKAQVSNEDCLQVE 27-41 KAQVSNEDCLQVENC 29-43 QVSNEDCLQVENCTQ 31-45SNEDCLQVENCTQLG 33-47 EDCLQVENCTQLGEQ 35-49 CLQVENCTQLGEQCW 37-51QVENCTQLGEQCWTA 39-53 ENCTQLGEQCWTARI 41-55 CTQLGEQCWTARIRA 43-57QLGEQCWTARIRAVG 45-59 GEQCWTARIRAVGLL 47-61 QCWTARIRAVGLLTV 49-63WTARIRAVGLLTVIS 51-65 ARIRAVGLLTVISKG 53-67 IRAVGLLTVISKGCS 55-69AVGLLTVISKGCSLN 57-71 GLLTVISKGCSLNCV 59-73 LTVISKGCSLNCVDD 61-75VISKGCSLNCVDDSQ 63-77 SKGCSLNCVDDSQDY 65-79 GCSLNCVDDSQDYYV 67-81SLNCVDDSQDYYVGK 69-83 NCVDDSQDYYVGKKN 71-85 VDDSQDYYVGKKNIT 73-87DSQDYYVGKKNITCC 75-89 QDYYVGKKNITCCDT 77-91 YYVGKKNITCCDTDL 79-93VGKKNITCCDTDLCN 81-95 KKNITCCDTDLCNAS 83-97 NITCCDTDLCNASGA 85-99TCCDTDLCNASGAHA  87-101 CDTDLCNASGAHALQ  89-103 TDLCNASGAHALQPA  91-105LCNASGAHALQPAAA  93-107 NASGAHALQPAAAIL  95-109 SGAHALQPAAAILAL  97-111AHALQPAAAILALLP  99-113 ALQPAAAILALLPAL 101-115 QPAAAILALLPALGL 103-117AAAILALLPALGLLL 105-119 AILALLPALGLLLWG 107-121 LALLPALGLLLWGPG 109-123LLPALGLLLWGPGQL 111-125 PALGLLLWGPGQL

TABLE 27 IFNγ T cell responses induced by the single antigen (Group 1:Ad-PSMA; Group 2: Ad-PSA; Group 3: Ad-PSCA; Group 4: mix of Ad-PSMA,Ad-PSA and Ad- PSCA) or triple antigen expressing adenovirus vectors(Group 4: Ad- 733; Group 6: Ad- 734; Group 7: Ad-735; Group 8: Ad-796and Group 9: Ad- 809) after adenovirus prime with anti-CTLA-4 analyzedby ELISPOT assay. Response to PSMA animal ID peptides 1 2 3 4 5 6 7 8Group 1 2356 988 1505 335 501 2145 NA NA No. 2 342 1776 154 329 158 438 321 NA 3 0 1276 40 126 20 0 NA NA 4 304 1198 774 2007 1277 1310 11592774 5 943 2670 2757 780 1082 2251 1566 544 6 472 2092 4248 1369 17602964 1447 263 7 2161 2202 939 869 3513 1654 3424 900 8 1166 799 2566 6631043 497 1334 560 9 1621 3247 2031 980 2942 1882 1918 3805 Response toPSA animal ID peptides 1 2 3 4 5 6 7 8 Group 1 0 0 0 48 0 42 NA NA No. 21419 1426 298 1223 1346 1120 1694 NA 3 6 462 91 0 77 0 NA NA 4 790 10931611 790 186 783 2016 1964 5 101 510 955 665 336 1512 1052 119 6 236 6732155 724 504 1600  930 83 7 0 1086 494 663 2265 117 1712 84 8 1893 20601490 1759 2352 1700 2232 1326 9 1193 1432 207 1738 1886 949  492 1940Response to PSCA animal ID peptides 1 2 3 4 5 6 7 8 Group 1 795 425 8741069 219 203 NA NA No. 2 669 713 391 199 164 560 461 NA 3 510 1234 10991115 1194 339 NA NA 4 778 528 680 1101 165 531 1175  1009  5 378 10611161 143 71 756 766 204 6 118 380 1190 403 829 1225 148 261 7 615 1141794 564 1175 490 856 204 8 968 1136 745 290 550 976 955 841 9 929 4341150 745 1120 246 1195  970

TABLE 28 IFNγ T cell responses induced by the single antigen (Group 1:PSMA; Group 2: PSA; Group 3: PSCA; Group 4: mix of PSMA, PSA and PSCA)or triple antigen expressing vectors (Groups 5-9) after adenovirus primewith anti-CTLA-4 and DNA electroporation boost immunizations analyzed byELISPOT assay. Response to PSMA animal ID peptides 1 2 3 4 5 6 7 8 Group1 1327 1535 1643 535 1506 1267 NA NA No. 2 15 266 26 191 10 46 1305 NA 30 445 5 75 4 6 NA NA 4 365 675 731 1134 244 714 999 1683 5 270 1623 2254626 860 2245 1453 1046 6 541 1151 2923 1094 1061 1746 691 489 7 11831183 1453 1649 2844 1470 2321 991 8 486 69 399 216 351 758 416 1389 91430 2631 2015 475 1368 1826 1851 3141 Response to PSA animal IDpeptides 1 2 3 4 5 6 7 8 Group 1 0 0 0 1 0 26 NA NA No. 2 1883 1236 1574393 461 941 1565  NA 3 33 30 9 13 8 11 NA NA 4 571 1129 1180 210 88 274924 360 5 50 1255 1344 628 210 638 948 1161 6 88 228 1390 489 1006 908683 51 7 0 211 321 156 1509 56 199 85 8 414 611 85 105 544 1080 331 18839 434 821 556 343 1160 510 144 1115 Response to PSCA animal ID peptides1 2 3 4 5 6 7 8 Group 1 615 799 533 74 258 61 NA NA No. 2 194 170 133133 8 66 405 NA 3 819 1071 873 839 1045 724 NA NA 4 543 506 664 470 70673 761 1235 5 154 455 1218 109 218 1094 285 569 6 56 293 603 506 745911  63 165 7 429 298 939 589 1226 263 803 451 8 279 214 871 61 144 511193 963 9 379 191 1196 73 699 198 616 836

Example 20 Reduction of STAT3 Phosphorylation by Sunitinib

The following example is provided to illustrate the capability ofsunitinib to directly inhibit the phosphorylation of STAT3 (signaltransducer of activator of transcription 3), a key mediator of immunesuppression in the spleen.

Study Procedure.

The acute effect of Sutent on the phosphorylation status of STAT3 in thespleen was investigated in a subcutaneous tumor mouse model to evaluatethe direct immunomodulatory effects of the compound. Briefly, 10-12 weekold female BALB/neuT mice were implanted with 1e6 TUBO cellssubcutaneously in the right flank. After forty one days post tumorimplant, Sutent was given by oral gavage at 20 mg/kg. At 0, 1, 3, 7 and24 hrs post Sutent dosing, three animals per timepoint were sacrificedunder IAUCUC guidelines and spleens were immediately snap frozen inliquid nitrogen to preserve the phosphorylation status. Spleens fromfemale BALB/c mice were snap frozen to use as healthy mice controls.

STAT3 Assay Procedures.

Snap frozen spleens were homogenized at 100 mg tissue per 500 μL lysisbuffer (70 mM NaCl, 50 mM β-glycerol phosphate, 10 mM HEPES, 1% TritonX-100, 100 mM Na₃VO₄, 100 mM PMSF, 1 mg/mL leupeptin) using a polytrontissue homogenizer. Resulting digests were centrifuged at 10,000 g for15 minutes. The supernatant was isolated and protein concentrations weredetermined using BCA protein assay kit (Pierce, Rockford, Ill.). Fortymicrograms of protein were added to each well of either a total STAT3(eBioscience, cat no. 85-86101-11) or phosphor-STAT3 (eBioscience, catno. 85-86102-11) ELISA Kit. Relative levels of either protein werecompared with standards provided in the kit and with standards purchasedindependently (Signaling Technologies, cat no. 9333-S).

Results.

Table 29 shows the result of a representative STAT assay that evaluatesthe effect of Sutent on the phosphorylation status of STAT3 in thespleen. Both spleen extracts from healthy or tumor bearing miceexhibited similar levels of STAT3 protein by ELISA (Total STAT3).However, compared to healthy BALB/c, the extracts from tumor bearingmice had significantly higher levels of phosphorylated STAT3 (Student'sT-test, P<0.001). The phosphorylation levels rapidly decreased to levelssimilar to healthy animals only 1 hr after Sutent treatment andmaintained at lower levels than the untreated mice up to 7 hrs. At 24hrs the phosphorylation levels of STAT3 completely recovered to levelsbefore Sutent treatment. The phosphorylation kinetics mirrors the levelsof circulating Sutent in the blood. The rapid response of STAT3phosphorylation in the spleen reflecting the pharmacokinetic profile ofSutent suggests a direct immunomodulatory function of Sutent in tumorbearing animals.

TABLE. 29 The relative levels of phosphorylated STAT3 and total STAT3from healthy BALB/c and tumor bearing BALB/neuT mice before or afterSutent treatment at multiple time points. Time Phospho-STAT3 Total STAT3Point, Individual Individual Strain hrs values Mean SEM values Mean SEMBalb/c 0 0.11 0.09 0.01 0.12 0.13 0.00 0.08 0.12 0.09 0.14 Tumor 0 1.081.31 0.12 0.08 0.09 0.01 bearing 1.38 0.11 BALB/neuT 1.46 NA 1 0.26 0.230.02 0.12 0.09 0.01 0.25 0.08 0.19 0.08 3 0.19 0.19 0.02 0.15 0.13 0.020.15 0.09 0.22 0.16 7 0.19 0.27 0.04 0.10 0.08 0.01 0.31 0.07 0.29 0.0824 1.54 1.44 0.07 0.08 0.08 0.00 1.30 0.08 1.47 0.08

RAW SEQUENCE LISTING SEQ ID NO: 1. AMINO ACID SEQUENCE OF THE FULLLENGTH HUMAN PSMAMWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSE VA SEQ IDNO: 2. NUCLEOTIDE SEQUENCE ENCODING THE FULL LENGTH HUMAN PSMA OF SEQ IDNO: 1atgtggaatctccttcacgaaaccgactcggctgtggccaccgcgcgccgcccgcgctggctgtgcgctggggcgctggtgctggcgggtggcttctttctcctcggcttcctcttcgggtggtttataaaatcctccaatgaagctactaacattactccaaagcataatatgaaagcatttttggatgaattgaaagctgagaacatcaagaagttcttatataattttacacagataccacatttagcaggaacagaacaaaactttcagcttgcaaagcaaattcaatcccagtggaaagaatttggcctggattctgttgagctagcacattatgatgtcctgttgtcctacccaaataagactcatcccaactacatctcaataattaatgaagatggaaatgagattttcaacacatcattatttgaaccacctcctccaggatatgaaaatgtttcggatattgtaccacctttcagtgctttctctcctcaaggaatgccagagggcgatctagtgtatgttaactatgcacgaactgaagacttctttaaattggaacgggacatgaaaatcaattgctctgggaaaattgtaattgccagatatgggaaagttttcagaggaaataaggttaaaaatgcccagctggcaggggccaaaggagtcattctctactccgaccctgctgactactttgctcctggggtgaagtcctatccagatggttggaatcttcctggaggtggtgtccagcgtggaaatatcctaaatctgaatggtgcaggagaccctctcacaccaggttacccagcaaatgaatatgcttataggcgtggaattgcagaggctgttggtcttccaagtattcctgttcatccaattggatactatgatgcacagaagctcctagaaaaaatgggtggctcagcaccaccagatagcagctggagaggaagtctcaaagtgccctacaatgttggacctggctttactggaaacttttctacacaaaaagtcaagatgcacatccactctaccaatgaagtgacaagaatttacaatgtgataggtactctcagaggagcagtggaaccagacagatatgtcattctgggaggtcaccgggactcatgggtgtttggtggtattgaccctcagagtggagcagctgttgttcatgaaattgtgaggagctttggaacactgaaaaaggaagggtggagacctagaagaacaattttgtttgcaagctgggatgcagaagaatttggtcttcttggttctactgagtgggcagaggagaattcaagactccttcaagagcgtggcgtggcttatattaatgctgactcatctatagaaggaaactacactctgagagttgattgtacaccgctgatgtacagcttggtacacaacctaacaaaagagctgaaaagccctgatgaaggctttgaaggcaaatctctttatgaaagttggactaaaaaaagtccttccccagagttcagtggcatgcccaggataagcaaattgggatctggaaatgattttgaggtgttcttccaacgacttggaattgcttcaggcagagcacggtatactaaaaattgggaaacaaacaaattcagcggctatccactgtatcacagtgtctatgaaacatatgagttggtggaaaagttttatgatccaatgtttaaatatcacctcactgtggcccaggttcgaggagggatggtgtttgagctagccaattccatagtgctcccttttgattgtcgagattatgctgtagttttaagaaagtatgctgacaaaatctacagtatttctatgaaacatccacaggaaatgaagacatacagtgtatcatttgattcacttttttctgcagtaaagaattttacagaaattgcttccaagttcagtgagagactccaggactttgacaaaagcaacccaatagtattaagaatgatgaatgatcaactcatgtttctggaaagagcatttattgatccattagggttaccagacaggcctttttataggcatgtcatctatgctccaagcagccacaacaagtatgcaggggagtcattcccaggaatttatgatgctctgtttgatattgaaagcaaagtggacccttccaaggcctggggagaagtgaagagacagatttatgttgcagccttcacagtgcaggcagctgcagagactttgagtgaagtagcc SEQ ID NO: 3.AMINO ACID SEQUENCE OF PSMA SHUFFLED ANTIGEN 1MASARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSSEATNISPQHNVKAFLDEMKAENIKKFLYLFTQIPHLAGTEQNFQLAKQIQAEWKEFGLDSVELAHYDVLLSYPNETHPNYISIIDEDGNEIFNTSLFEPPPPGYENISDVVPPYSAFSPQGMPEGDLVYVNYARTEDFFKLERELKINCSGKILIARYGKVFRGNKVKNAQLAGAKGIILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNVLNLNGAGDPLTPGYPANEYAYRRELAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWKGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTIRGAVEPDRYVILGGHRDAWVFGGIDPQSGAAVVHEIVRSFGTLKKKGWRPRRTIIFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVYNLTKELQSPDEGFEGKSLYESWTKKSPSPEFSGVPRINKLGSGNDFEVFFQRLGIASGRARYTKNWKTNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGLVFELADSIVLPFDCQDYAVVLRKYADKIYNLAMKHPEELKTYSVSFDSLFSAVKNFTEIASKFNQRLQDFDKNNPLLVRMLNDQLMFLERAFVDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA SEQ ID NO: 4. NUCLEOTIDESEQUENCE ENCODING AMINO ACID SEQUENCE OF PSMA SHUFFLED ANTIGEN 1 OF SEQID NO: 3atggctagcgccagacggcccagatggctgtgcgccggagccctggtgctggccggaggattcttcctgctgggcttcctgttcggctggttcatcaagagcagcagcgaggccaccaacatcagcccccagcacaacgtgaaggcctttctggacgagatgaaggccgagaacatcaagaagtttctgtacctgttcacccagatcccccacctggccggcaccgagcagaacttccagctggccaagcagattcaggctgagtggaaagagttcggcctggacagcgtggagctggcccactacgacgtgctgctgtcctaccccaacgagacacaccccaactacatcagcatcatcgacgaggacggcaacgagattttcaacaccagcctgttcgagccccctccccctggctacgagaacatctccgacgtggtgcccccctacagcgccttcagccctcagggaatgcctgaaggcgacctggtgtacgtgaactacgcccggaccgaggacttcttcaagctggaacgggagctgaagatcaactgcagcggcaagatcctgatcgccagatacggcaaggtgttccggggcaacaaagtgaagaacgcacagctggctggagccaagggcatcatcctgtacagcgaccccgccgactacttcgcccctggcgtgaagtcctaccctgacggctggaacctgcctggcggcggagtgcagcggggcaacgtgctgaacctgaacggagccggcgaccctctgaccccaggctaccccgccaacgagtacgcctaccggcgggagctggccgaagccgtgggcctgcccagcatccccgtgcaccccatcggctactacgacgcccagaaactgctggaaaagatgggcggcagcgcccctcccgacagcagctggaagggcagcctgaaggtgccctacaacgtgggccctggcttcaccggcaacttcagcacccagaaagtgaagatgcacatccacagcaccaacgaagtgacccggatctacaacgtgatcggcaccatcagaggcgccgtggagcccgacagatacgtgatcctgggcggccaccgggacgcctgggtgttcggcggcatcgacccccagagcggagccgccgtggtgcacgagatcgtgcggagcttcggcaccctgaagaagaagggctggcggcccagacggaccatcatcttcgccagctgggacgccgaggaattcggactgctgggctctaccgagtgggccgaggaaaacagcagactgctgcaggaacggggcgtcgcctacatcaacgccgacagctccatcgagggcaactacaccctgcgggtggactgcacccccctgatgtacagcctggtgtacaacctgaccaaagagctgcagagccccgacgagggcttcgagggcaagagcctgtacgagagctggaccaagaagtcccccagccccgagttcagcggcgtgccccggatcaacaagctgggcagcggcaacgacttcgaggtgttcttccagaggctgggcattgccagcggcagagcccggtacaccaagaactggaaaaccaacaagttctccggctaccccctgtaccacagcgtgtacgagacatacgaactggtggagaagttctacgaccccatgttcaagtaccacctgaccgtggcccaggtccggggagggctggtgttcgaactggccgacagcatcgtgctgcccttcgactgccaggactatgctgtggtgctgcggaagtacgccgacaaaatctacaacctggccatgaagcaccccgaggaactgaaaacctacagcgtgtccttcgacagcctgttcagcgccgtgaagaacttcaccgagatcgccagcaagttcaaccagcggctgcaggacttcgacaagaacaaccccctgctggtccggatgctgaacgaccagctgatgttcctggaacgggccttcgtggaccccctgggcctgcctgaccggcccttctaccggcacgtgatctatgcccccagcagccacaacaagtacgctggcgagagcttccccggcatctacgatgccctgttcgacatcgagagcaaggtggaccccagcaaggcctggggcgaagtgaagcggcagatatacgtggccgccttcacagtgcaggccgctgccgagacactgagcgaggtggcc SEQ ID NO: 5.AMINO ACID SEQUENCE OF PSMA SHUFFLED ANTIGEN 2MASARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSSEATNITPQHNVKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFELAKQIQAQWKEFGLDSVELSHYDVLLSYPNETHPNYISIIDEDGNEIFNTSLFEPPPPGYENISDVVPPYSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKILIARYGKVFRGNKVKNAQLAGAKGIILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNVLNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGAAPPDSSWKGSLQVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLKGAVEPDRYVILGGHRDAWVFGGIDPQSGAAVVHEIVRSFGTLKKKGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVYNLTKELQSPDEGFEGKSLFDSWTEKSPSPEFSGLPRISKLGSGNDFEVFFQRLGIASGRARYTKDWKTSKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGIVFELANSVVLPFDCQDYAVVLKKYADKIYNISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFNQRLQDFDKNNPILLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA SEQ ID NO: 6. NUCLEOTIDESEQUENCE ENCODING AMINO ACID SEQUENCE OF PSMA SHUFFLED ANTIGEN 2 OF SEQID NO: 5atggctagcgccagacggcccagatggctgtgtgctggcgccctggtgctggctggcggctttttcctgctgggcttcctgttcggctggttcatcaagagcagcagcgaggccaccaacatcaccccccagcacaacgtgaaggcctttctggacgagctgaaggccgagaatatcaagaagttcctgtacaacttcacccagatcccccacctggccggcaccgagcagaacttcgagctggccaagcagatccaggcccagtggaaagagttcggcctggacagcgtggaactgagccactacgacgtgctgctgagctaccccaacgagacacaccccaactacatcagcatcatcgacgaggacggcaacgagattttcaacaccagcctgttcgagccccctccacccggctacgagaacatcagcgacgtggtgcccccctacagcgcattcagtccacagggaatgcccgagggcgacctggtgtacgtgaactacgcccggaccgaggacttcttcaagctggaacgggacatgaagatcaactgcagcggcaagatcctgatcgccagatacggcaaggtgttccggggcaacaaagtgaagaacgcccagctggcaggcgccaagggcatcatcctgtacagcgaccccgccgactacttcgcccctggcgtgaagtcctaccccgacggctggaacctgcctggcggcggagtgcagaggggcaacgtgctgaacctgaacggcgctggcgaccctctgacccctggctaccccgccaacgagtacgcctacagacggggaatcgccgaggccgtgggcctgcctagcatccctgtgcaccccatcggctactacgacgcccagaaactgctggaaaagatgggcggagccgcccctcccgacagctcttggaagggcagcctgcaggtcccctacaacgtgggccctggcttcaccggcaacttcagcacccagaaagtgaagatgcacatccacagcaccaacgaagtgacccggatctacaacgtgatcggcaccctgaagggcgccgtggaacccgacagatacgtgatcctgggcggccaccgggacgcctgggtgttcggaggcatcgaccctcagagcggcgctgccgtggtgcacgagatcgtgcggagcttcggcacactgaagaagaagggctggcggcccagacggaccatcctgttcgccagctgggacgccgaggaattcggcctgctgggcagcaccgagtgggccgaggaaaacagtcggctgctgcaggaacggggcgtcgcctacatcaacgccgacagcagcatcgagggcaactacaccctgcgggtggactgcacccccctgatgtacagcctggtgtacaacctgaccaaagagctgcagagccccgacgagggcttcgagggcaagtccctgttcgactcctggaccgagaagtcccccagccccgagttcagcggcctgcccagaatcagcaagctgggcagcggcaacgacttcgaggtgttcttccagcggctgggaatcgccagcggcagagcccggtacaccaaggactggaaaaccagcaagttctccggctaccccctgtaccacagcgtgtacgagacatacgagctggtggaaaagttctacgaccccatgttcaagtaccacctgaccgtggcccaggtccgaggcggcatcgtgttcgaactggccaacagcgtggtgctgccattcgattgtcaggactacgccgtggtgctgaagaagtacgccgacaaaatctacaacatcagcatgaagcacccccaggaaatgaaaacctacagcgtgtccttcgacagcctgttcagcgccgtgaagaatttcaccgagatcgcctccaagttcaaccagagactgcaggacttcgacaagaacaaccccatcctgctgcggatgatgaacgaccagctgatgttcctggaacgggccttcatcgaccccctgggcctgcccgaccggcccttttaccggcacgtgatctatgcccccagcagccacaacaaatacgccggcgagagtttccccggcatctacgatgccctgttcgatatcgagagcaaggtggaccccagcaaggcctggggcgaagtgaagcggcagatttacgtggccgcattcacagtgcaggctgctgccgagacactgagcgaggtggcc SEQ ID NO: 7. AMINOACID SEQUENCE OF PSMA SHUFFLED ANTIGEN 3MASARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSAQKLKLHIHSNTKVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRTFGTLKKKGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLLHSLVYNLTKELKSPDEGFEGKSLYESWTKKSPSPELSGLPRISKLGSGNDFEVFFQRLGISSGRARYTKDWKTSKFSSYPLYHSIYETYELVVKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVALKNHAENLYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA SEQ ID NO: 8. NUCLEOTIDESEQEUNCE ENCODING AMINO ACID SEQUENCE OF PSMA SHUFFLED ANTIGEN 3 OF SEQID NO: 7atggctagcgccagacggcccagatggctgtgtgctggcgccctggtgctggctggcggctttttcctgctgggcttcctgttcggctggttcatcaagagcagcaacgaggccaccaacatcacccccaagcacaacatgaaggcctttctggacgagctgaaggccgagaatatcaagaagttcctgtacaacttcacccagatcccccacctggccggcaccgagcagaacttccagctggccaagcagatccagagccagtggaaagagttcggcctggacagcgtggaactggcccactacgacgtgctgctgagctaccccaacaagacccaccccaactacatcagcatcatcaacgaggacggcaacgagattttcaacaccagcctgttcgagccccctccacccggctacgagaacgtgtccgacatcgtgcccccattcagcgcattcagtccacagggaatgcccgagggcgacctggtgtacgtgaactacgcccggaccgaggacttcttcaagctggaacgggacatgaagatcaactgcagcggcaagatcgtgatcgccagatacggcaaggtgttccggggcaacaaagtgaagaacgcccagctggcaggcgccaagggcgtgatcctgtatagcgaccccgccgactacttcgcccctggcgtgaagtcctaccccgacggctggaacctgcctggcggcggagtgcagcggggcaacatcctgaacctgaacggcgctggcgaccccctgacccctggctatcccgccaacgagtacgcctacagacggggaatcgccgaggccgtgggcctgcctagcatccctgtgcaccccatcggctactacgacgcccagaaactgctggaaaagatgggcggcagcgcccctcccgatagctcttggagaggcagcctgaaggtgccctacaacgtgggccctggcttcaccggcaacttcagcgcccagaagctgaagctgcacatccacagcaacaccaaagtgacccggatctacaacgtgatcggcaccctgagaggcgccgtggaacccgacagatacgtgatcctgggcggccaccgggacagctgggtgttcggcggcatcgaccctcagtctggcgccgctgtggtgcacgagatcgtgcggacctttggcaccctgaagaagaagggctggcggcccagacggaccatcctgttcgccagctgggacgccgaggaattcggcctgctgggcagcaccgagtgggccgaggaaaacagtcggctgctgcaggaacggggcgtcgcctacatcaacgccgacagcagcatcgagggcaactacaccctgcgggtggactgcacccccctgctgcacagcctggtgtacaacctgaccaaagagctgaagtcccccgacgagggcttcgagggcaagagcctgtacgagagctggaccaagaagtcccccagccccgagctgagcggcctgcccagaatcagcaagctgggcagcggcaacgacttcgaggtgttcttccagcggctgggcatcagcagcggcagagcccggtacaccaaggactggaaaaccagcaagttcagcagctaccccctgtaccacagcatctacgagacatacgagctggtggtcaagttctacgaccccatgttcaagtaccacctgaccgtggcccaggtccgaggcggcatggtgttcgagctggccaacagcatcgtgctgcccttcgactgccgggactacgccgtggccctgaagaaccacgccgagaacctgtacagcatcagcatgaagcacccccaggaaatgaaaacctacagcgtgtccttcgacagcctgttcagcgccgtgaagaatttcaccgagatcgcctccaagttcagcgagcggctgcaggacttcgacaagagcaaccccatcgtgctgagaatgatgaacgaccagctgatgttcctggaacgggccttcatcgaccccctgggcctgcccgaccggcccttttaccggcacgtgatctatgcccccagcagccacaacaaatacgccggcgagagtttccccggcatctacgatgccctgttcgacatcgagagcaaggtggaccccagcaaggcctggggcgaagtgaagcggcagatttacgtggccgcattcacagtgcaggccgctgccgagacactgagcgaggtggcc SEQ ID NO: 9. AMINOACID SEQUENCE OF A MEMBRANE-BOUND PSMA ANTIGENMASARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA SEQ ID NO: 10.NUCLEOTIDE SEQEUNCE ENCODING AMINO ACID SEQUENCE OF THE MEMBRANE-BOUNDPSMA ANTIGEN OF SEQ ID NO: 9atggctagcgcgcgccgcccgcgctggctgtgcgctggggcgctggtgctggcgggtggcttctttctcctcggcttcctcttcgggtggtttataaaatcctccaatgaagctactaacattactccaaagcataatatgaaagcatttttggatgaattgaaagctgagaacatcaagaagttcttatataattttacacagataccacatttagcaggaacagaacaaaactttcagcttgcaaagcaaattcaatcccagtggaaagaatttggcctggattctgttgagctggcacattatgatgtcctgttgtcctacccaaataagactcatcccaactacatctcaataattaatgaagatggaaatgagattttcaacacatcattatttgaaccacctcctccaggatatgaaaatgtttcggatattgtaccacctttcagtgctttctctcctcaaggaatgccagagggcgatctagtgtatgttaactatgcacgaactgaagacttctttaaattggaacgggacatgaaaatcaattgctctgggaaaattgtaattgccagatatgggaaagttttcagaggaaataaggttaaaaatgcccagctggcaggggccaaaggagtcattctctactccgaccctgctgactactttgctcctggggtgaagtcctatccagatggttggaatcttcctggaggtggtgtccagcgtggaaatatcctaaatctgaatggtgcaggagaccctctcacaccaggttacccagcaaatgaatatgcttataggcgtggaattgcagaggctgttggtcttccaagtattcctgttcatccaattggatactatgatgcacagaagctcctagaaaaaatgggtggctcagcaccaccagatagcagctggagaggaagtctcaaagtgccctacaatgttggacctggctttactggaaacttttctacacaaaaagtcaagatgcacatccactctaccaatgaagtgacaagaatttacaatgtgataggtactctcagaggagcagtggaaccagacagatatgtcattctgggaggtcaccgggactcatgggtgtttggtggtattgaccctcagagtggagcagctgttgttcatgaaattgtgaggagctttggaacactgaaaaaggaagggtggagacctagaagaacaattttgtttgcaagctgggatgcagaagaatttggtcttcttggttctactgagtgggcagaggagaattcaagactccttcaagagcgtggcgtggcttatattaatgctgactcatctatagaaggaaactacactctgagagttgattgtacaccgctgatgtacagcttggtacacaacctaacaaaagagctgaaaagccctgatgaaggctttgaaggcaaatctctttatgaaagttggactaaaaaaagtccttccccagagttcagtggcatgcccaggataagcaaattgggatctggaaatgattttgaggtgttcttccaacgacttggaattgcttcaggcagagcacggtatactaaaaattgggaaacaaacaaattcagcggctatccactgtatcacagtgtctatgaaacatatgagttggtggaaaagttttatgatccaatgtttaaatatcacctcactgtggcccaggttcgaggagggatggtgtttgagctggccaattccatagtgctcccttttgattgtcgagattatgctgtagttttaagaaagtatgctgacaaaatctacagtatttctatgaaacatccacaggaaatgaagacatacagtgtatcatttgattcacttttttctgcagtaaagaattttacagaaattgcttccaagttcagtgagagactccaggactttgacaaaagcaacccaatagtattaagaatgatgaatgatcaactcatgtttctggaaagagcatttattgatccattagggttaccagacaggcctttttataggcatgtcatctatgctccaagcagccacaacaagtatgcaggggagtcattcccaggaatttatgatgctctgtttgatattgaaagcaaagtggacccttccaaggcctggggagaagtgaagagacagatttatgttgcagccttcacagtgcaggcagctgcagagactttgagtgaagtagcc SEQ ID NO: 11. AMINO ACID SEQUENCE OF A CYTOSOLICPSMA ANTIGEN MASKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA SEQ ID NO: 12. NUCLEOTIDE SEQEUNCE ENCODING AMINO ACIDSEQUENCE OF THE CYTOSOLIC PSMA ANTIGEN OF SEQ ID NO: 11atggctagcaaatcctccaatgaagctactaacattactccaaagcataatatgaaagcatttttggatgaattgaaagctgagaacatcaagaagttcttatataattttacacagataccacatttagcaggaacagaacaaaactttcagcttgcaaagcaaattcaatcccagtggaaagaatttggcctggattctgttgagctggcacattatgatgtcctgttgtcctacccaaataagactcatcccaactacatctcaataattaatgaagatggaaatgagattttcaacacatcattatttgaaccacctcctccaggatatgaaaatgtttcggatattgtaccacctttcagtgctttctctcctcaaggaatgccagagggcgatctagtgtatgttaactatgcacgaactgaagacttctttaaattggaacgggacatgaaaatcaattgctctgggaaaattgtaattgccagatatgggaaagttttcagaggaaataaggttaaaaatgcccagctggcaggggccaaaggagtcattctctactccgaccctgctgactactttgctcctggggtgaagtcctatccagatggttggaatcttcctggaggtggtgtccagcgtggaaatatcctaaatctgaatggtgcaggagaccctctcacaccaggttacccagcaaatgaatatgcttataggcgtggaattgcagaggctgttggtcttccaagtattcctgttcatccaattggatactatgatgcacagaagctcctagaaaaaatgggtggctcagcaccaccagatagcagctggagaggaagtctcaaagtgccctacaatgttggacctggctttactggaaacttttctacacaaaaagtcaagatgcacatccactctaccaatgaagtgacaagaatttacaatgtgataggtactctcagaggagcagtggaaccagacagatatgtcattctgggaggtcaccgggactcatgggtgtttggtggtattgaccctcagagtggagcagctgttgttcatgaaattgtgaggagctttggaacactgaaaaaggaagggtggagacctagaagaacaattttgtttgcaagctgggatgcagaagaatttggtcttcttggttctactgagtgggcagaggagaattcaagactccttcaagagcgtggcgtggcttatattaatgctgactcatctatagaaggaaactacactctgagagttgattgtacaccgctgatgtacagcttggtacacaacctaacaaaagagctgaaaagccctgatgaaggctttgaaggcaaatctctttatgaaagttggactaaaaaaagtccttccccagagttcagtggcatgcccaggataagcaaattgggatctggaaatgattttgaggtgttcttccaacgacttggaattgcttcaggcagagcacggtatactaaaaattgggaaacaaacaaattcagcggctatccactgtatcacagtgtctatgaaacatatgagttggtggaaaagttttatgatccaatgtttaaatatcacctcactgtggcccaggttcgaggagggatggtgtttgagctggccaattccatagtgctcccttttgattgtcgagattatgctgtagttttaagaaagtatgctgacaaaatctacagtatttctatgaaacatccacaggaaatgaagacatacagtgtatcatttgattcacttttttctgcagtaaagaattttacagaaattgcttccaagttcagtgagagactccaggactttgacaaaagcaacccaatagtattaagaatgatgaatgatcaactcatgtttctggaaagagcatttattgatccattagggttaccagacaggcctttttataggcatgtcatctatgctccaagcagccacaacaagtatgcaggggagtcattcccaggaatttatgatgctctgtttgatattgaaagcaaagtggacccttccaaggcctggggagaagtgaagagacagatttatgttgcagccttcacagtgcaggcagctgcagagactttgagtgaagtagcc SEQ ID NO: 13. AMINO ACID SEQUENCE OF A SECRETED PSMAANTIGEN MASETDTLLLWVLLLWVPGSTGDAAKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA SEQ ID NO: 14. NUCLEOTIDESEQUENCE ENCODING AMINO ACID SEQUENCE OF THE SECRETED PSMA ANTIGEN OFSEQ ID NO: 13atggctagcgaaaccgacactttgttgttgtgggtgcttttgctttgggtacccggatctactggtgatgctgctaaatcctccaatgaagctactaacattactccaaagcataatatgaaagcatttttggatgaattgaaagctgagaacatcaagaagttcttatataattttacacagataccacatttagcaggaacagaacaaaactttcagcttgcaaagcaaattcaatcccagtggaaagaatttggcctggattctgttgagctagcacattatgatgtcctgttgtcctacccaaataagactcatcccaactacatctcaataattaatgaagatggaaatgagattttcaacacatcattatttgaaccacctcctccaggatatgaaaatgtttcggatattgtaccacctttcagtgctttctctcctcaaggaatgccagagggcgatctagtgtatgttaactatgcacgaactgaagacttctttaaattggaacgggacatgaaaatcaattgctctgggaaaattgtaattgccagatatgggaaagttttcagaggaaataaggttaaaaatgcccagctggcaggggccaaaggagtcattctctactccgaccctgctgactactttgctcctggggtgaagtcctatccagatggttggaatcttcctggaggtggtgtccagcgtggaaatatcctaaatctgaatggtgcaggagaccctctcacaccaggttacccagcaaatgaatatgcttataggcgtggaattgcagaggctgttggtcttccaagtattcctgttcatccaattggatactatgatgcacagaagctcctagaaaaaatgggtggctcagcaccaccagatagcagctggagaggaagtctcaaagtgccctacaatgttggacctggctttactggaaacttttctacacaaaaagtcaagatgcacatccactctaccaatgaagtgacaagaatttacaatgtgataggtactctcagaggagcagtggaaccagacagatatgtcattctgggaggtcaccgggactcatgggtgtttggtggtattgaccctcagagtggagcagctgttgttcatgaaattgtgaggagctttggaacactgaaaaaggaagggtggagacctagaagaacaattttgtttgcaagctgggatgcagaagaatttggtcttcttggttctactgagtgggcagaggagaattcaagactccttcaagagcgtggcgtggcttatattaatgctgactcatctatagaaggaaactacactctgagagttgattgtacaccgctgatgtacagcttggtacacaacctaacaaaagagctgaaaagccctgatgaaggctttgaaggcaaatctctttatgaaagttggactaaaaaaagtccttccccagagttcagtggcatgcccaggataagcaaattgggatctggaaatgattttgaggtgttcttccaacgacttggaattgcttcaggcagagcacggtatactaaaaattgggaaacaaacaaattcagcggctatccactgtatcacagtgtctatgaaacatatgagttggtggaaaagttttatgatccaatgtttaaatatcacctcactgtggcccaggttcgaggagggatggtgtttgagctagccaattccatagtgctcccttttgattgtcgagattatgctgtagttttaagaaagtatgctgacaaaatctacagtatttctatgaaacatccacaggaaatgaagacatacagtgtatcatttgattcacttttttctgcagtaaagaattttacagaaattgcttccaagttcagtgagagactccaggactttgacaaaagcaacccaatagtattaagaatgatgaatgatcaactcatgtttctggaaagagcatttattgatccattagggttaccagacaggcctttttataggcatgtcatctatgctccaagcagccacaacaagtatgcaggggagtcattcccaggaatttatgatgctctgtttgatattgaaagcaaagtggacccttccaaggcctggggagaagtgaagagacagatttatgttgcagccttcacagtgcaggcagctgcagagactttgagtgaagtagccSEQ ID NO: 15. AMINO ACID SEQUENCE OF THE FULL LENGTH HUMAN PSAMASWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP SEQ ID NO: 16. NUCLEOTIDE SEQUENCEENCODING AMINO ACID SEQUENCE OF THE FULL LENGTH HUMAN PSA OF SEQ ID NO:15atggctagctgggtcccggttgtcttcctcaccctgtccgtgacgtggattggcgctgcgcccctcatcctgtctcggattgtgggaggctgggagtgcgagaagcattcccaaccctggcaggtgcttgtggcctctcgtggcagggcagtctgcggcggtgttctggtgcacccccagtgggtcctcacagctgcccactgcatcaggaacaaaagcgtgatcttgctgggtcggcacagcttgtttcatcctgaagacacaggccaggtatttcaggtcagccacagcttcccacacccgctctacgatatgagcctcctgaagaatcgattcctcaggccaggtgatgactccagccacgacctcatgctgctccgcctgtcagagcctgccgagctcacggatgctgtgaaggtcatggacctgcccacccaggagccagcactggggaccacctgctacgcctcaggctggggcagcattgaaccagaggagttcttgaccccaaagaaacttcagtgtgtggacctccatgttatttccaatgacgtgtgtgcgcaagttcaccctcagaaggtgaccaagttcatgctgtgtgctggacgctggacagggggcaaaagcacctgctcgggtgattctgggggcccacttgtctgtaatggtgtgcttcaaggtatcacgtcatggggcagtgaaccatgtgccctgcccgaaaggccttccctgtacaccaaggtggtgcattaccggaagtggatcaaggacaccatcgtggccaaccccSEQ ID NO: 17. AMINO ACID SEQUENCE OF A CYTOSOLIC PSA ANTIGENMASIVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVV HYRKWIKDTIVANPSEQ ID NO: 18. NUCLEOTIDE SEQEUNCE ENCODING AMINO ACID SEQUENCE OF THECYTOSOLIC PSA ANTIGEN OF SEQ ID NO: 17atggctagcattgtgggaggctgggagtgcgagaagcattcccaaccctggcaggtgcttgtggcctctcgtggcagggcagtctgcggcggtgttctggtgcacccccagtgggtcctcacagctgcccactgcatcaggaacaaaagcgtgatcttgctgggtcggcacagcttgtttcatcctgaagacacaggccaggtatttcaggtcagccacagcttcccacacccgctctacgatatgagcctcctgaagaatcgattcctcaggccaggtgatgactccagccacgacctcatgctgctccgcctgtcagagcctgccgagctcacggatgctgtgaaggtcatggacctgcccacccaggagccagcactggggaccacctgctacgcctcaggctggggcagcattgaaccagaggagttcttgaccccaaagaaacttcagtgtgtggacctccatgttatttccaatgacgtgtgtgcgcaagttcaccctcagaaggtgaccaagttcatgctgtgtgctggacgctggacagggggcaaaagcacctgctcgggtgattctgggggcccacttgtctgtaatggtgtgcttcaaggtatcacgtcatggggcagtgaaccatgtgccctgcccgaaaggccttccctgtacaccaaggtggtgcattaccggaagtggatcaaggacaccatcgtggccaacccc SEQ ID NO: 19. AMINO ACID SEQUENCE OF A MEMBRANE-BOUND PSA ANTIGENMASARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPGIVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP SEQ ID NO: 20.NUCLEOTIDE SEQUENCE ENCODING AMINO ACID SEQUENCE OF THE MEMBRANE-BOUNDPSA ANTIGEN OF SEQ ID NO: 19atggctagcgcgcgccgcccgcgctggctgtgcgctggggcgctggtgctggcgggtggcttctttctcctcggcttcctcttcgggtggtttataaaatcctccaatgaagctactaacattactccaggaattgtgggaggctgggagtgcgagaagcattcccaaccctggcaggtgcttgtggcctctcgtggcagggcagtctgcggcggtgttctggtgcacccccagtgggtcctcacagctgcccactgcatcaggaacaaaagcgtgatcttgctgggtcggcacagcttgtttcatcctgaagacacaggccaggtatttcaggtcagccacagcttcccacacccgctctacgatatgagcctcctgaagaatcgattcctcaggccaggtgatgactccagccacgacctcatgctgctccgcctgtcagagcctgccgagctcacggatgctgtgaaggtcatggacctgcccacccaggagccagcactggggaccacctgctacgcctcaggctggggcagcattgaaccagaggagttcttgaccccaaagaaacttcagtgtgtggacctccatgttatttccaatgacgtgtgtgcgcaagttcaccctcagaaggtgaccaagttcatgctgtgtgctggacgctggacagggggcaaaagcacctgctcgggtgattctgggggcccacttgtctgtaatggtgtgcttcaaggtatcacgtcatggggcagtgaaccatgtgccctgcccgaaaggccttccctgtacaccaaggtggtgcattaccggaagtggatcaaggacaccatcgtggccaacccctga SEQ ID NO: 21. AMINO ACIDSEQUENCE OF THE FULL LENGTH HUMAN PSCAMASKAVLLALLMAGLALQPGTALLCYSCKAQVSNEDCLQVENCTQLGEQCWTARIRAVGLLTVISKGCSLNCVDDSQDYYVGKKNITCCDTDLCNASGAHALQPAAAILALLPAL GLLLWGPGQLSEQ ID NO: 22. NUCLEOTIDE SEQUENCE ENCODING AMINO ACID SEQUENCE OF THEFULL LENGTH HUMAN PSCA OF SEQ ID NO: 21atggctagcaaggctgtgctgcttgccctgttgatggcaggcttggccctgcagccaggcactgccctgctgtgctactcctgcaaagcccaggtgagcaacgaggactgcctgcaggtggagaactgcacccagctgggggagcagtgctggaccgcgcgcatccgcgcagttggcctcctgaccgtcatcagcaaaggctgcagcttgaactgcgtggatgactcacaggactactacgtgggcaagaagaacatcacgtgctgtgacaccgacttgtgcaacgccagcggggcccatgccctgcagccggctgccgccatccttgcgctgctccctgcactcggcctgctgctctggggacccggccagcta SEQ IDNO: 23. NUCLEOTIDE SEQUENCE OF PLASMID 5166GGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCAAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGGTCGACAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACTCACCGTCCGGATCTCAGCAAGCAGGTATGTACTCTCCAGGGTGGGCCTGGCTTCCCCAGTCAAGACTCCAGGGATTTGAGGGACGCTGTGGGCTCTTCTCTTACATGTACCTTTTGCTTGCCTCAACCCTGACTATCTTCCAGGTCAGGATCCCAGAGTCAGGGGTCTGTATTTTCCTGCTGGTGGCTCCAGTTCAGGAACAGTAAACCCTGCTCCGAATATTGCCTCTCACATCTCGTCAATCTCCGCGAGGACTGGGGACCCTGTGACGAACATGGCTAGCGCGCGCCGCCCGCGCTGGCTGTGCGCTGGGGCGCTGGTGCTGGCGGGTGGCTTCTTTCTCCTCGGCTTCCTCTTCGGGTGGTTTATAAAATCCTCCAATGAAGCTACTAACATTACTCCAAAGCATAATATGAAAGCATTTTTGGATGAATTGAAAGCTGAGAACATCAAGAAGTTCTTATATAATTTTACACAGATACCACATTTAGCAGGAACAGAACAAAACTTTCAGCTTGCAAAGCAAATTCAATCCCAGTGGAAAGAATTTGGCCTGGATTCTGTTGAGCTGGCACATTATGATGTCCTGTTGTCCTACCCAAATAAGACTCATCCCAACTACATCTCAATAATTAATGAAGATGGAAATGAGATTTTCAACACATCATTATTTGAACCACCTCCTCCAGGATATGAAAATGTTTCGGATATTGTACCACCTTTCAGTGCTTTCTCTCCTCAAGGAATGCCAGAGGGCGATCTAGTGTATGTTAACTATGCACGAACTGAAGACTTCTTTAAATTGGAACGGGACATGAAAATCAATTGCTCTGGGAAAATTGTAATTGCCAGATATGGGAAAGTTTTCAGAGGAAATAAGGTTAAAAATGCCCAGCTGGCAGGGGCCAAAGGAGTCATTCTCTACTCCGACCCTGCTGACTACTTTGCTCCTGGGGTGAAGTCCTATCCAGATGGTTGGAATCTTCCTGGAGGTGGTGTCCAGCGTGGAAATATCCTAAATCTGAATGGTGCAGGAGACCCTCTCACACCAGGTTACCCAGCAAATGAATATGCTTATAGGCGTGGAATTGCAGAGGCTGTTGGTCTTCCAAGTATTCCTGTTCATCCAATTGGATACTATGATGCACAGAAGCTCCTAGAAAAAATGGGTGGCTCAGCACCACCAGATAGCAGCTGGAGAGGAAGTCTCAAAGTGCCCTACAATGTTGGACCTGGCTTTACTGGAAACTTTTCTACACAAAAAGTCAAGATGCACATCCACTCTACCAATGAAGTGACAAGAATTTACAATGTGATAGGTACTCTCAGAGGAGCAGTGGAACCAGACAGATATGTCATTCTGGGAGGTCACCGGGACTCATGGGTGTTTGGTGGTATTGACCCTCAGAGTGGAGCAGCTGTTGTTCATGAAATTGTGAGGAGCTTTGGAACACTGAAAAAGGAAGGGTGGAGACCTAGAAGAACAATTTTGTTTGCAAGCTGGGATGCAGAAGAATTTGGTCTTCTTGGTTCTACTGAGTGGGCAGAGGAGAATTCAAGACTCCTTCAAGAGCGTGGCGTGGCTTATATTAATGCTGACTCATCTATAGAAGGAAACTACACTCTGAGAGTTGATTGTACACCGCTGATGTACAGCTTGGTACACAACCTAACAAAAGAGCTGAAAAGCCCTGATGAAGGCTTTGAAGGCAAATCTCTTTATGAAAGTTGGACTAAAAAAAGTCCTTCCCCAGAGTTCAGTGGCATGCCCAGGATAAGCAAATTGGGATCTGGAAATGATTTTGAGGTGTTCTTCCAACGACTTGGAATTGCTTCAGGCAGAGCACGGTATACTAAAAATTGGGAAACAAACAAATTCAGCGGCTATCCACTGTATCACAGTGTCTATGAAACATATGAGTTGGTGGAAAAGTTTTATGATCCAATGTTTAAATATCACCTCACTGTGGCCCAGGTTCGAGGAGGGATGGTGTTTGAGCTGGCCAATTCCATAGTGCTCCCTTTTGATTGTCGAGATTATGCTGTAGTTTTAAGAAAGTATGCTGACAAAATCTACAGTATTTCTATGAAACATCCACAGGAAATGAAGACATACAGTGTATCATTTGATTCACTTTTTTCTGCAGTAAAGAATTTTACAGAAATTGCTTCCAAGTTCAGTGAGAGACTCCAGGACTTTGACAAAAGCAACCCAATAGTATTAAGAATGATGAATGATCAACTCATGTTTCTGGAAAGAGCATTTATTGATCCATTAGGGTTACCAGACAGGCCTTTTTATAGGCATGTCATCTATGCTCCAAGCAGCCACAACAAGTATGCAGGGGAGTCATTCCCAGGAATTTATGATGCTCTGTTTGATATTGAAAGCAAAGTGGACCCTTCCAAGGCCTGGGGAGAAGTGAAGAGACAGATTTATGTTGCAGCCTTCACAGTGCAGGCAGCTGCAGAGACTTTGAGTGAAGTAGCCTAAAGATCTGGGCCCTAACAAAACAAAAAGATGGGGTTATTCCCTAAACTTCATGGGTTACGTAATTGGAAGTTGGGGGACATTGCCACAAGATCATATTGTACAAAAGATCAAACACTGTTTTAGAAAACTTCCTGTAAACAGGCCTATTGATTGGAAAGTATGTCAAAGGATTGTGGGTCTTTTGGGCTTTGCTGCTCCATTTACACAATGTGGATATCCTGCCTTAATGCCTTTGTATGCATGTATACAAGCTAAACAGGCTTTCACTTTCTCGCCAACTTACAAGGCCTTTCTAAGTAAACAGTACATGAACCTTTACCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGGCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCCGGTCTGGAGCAAAGCTCATAGGAACTGACAATTCTGTCGTCCTCTCGCGGAAATATACATCGTTTCGATCTACGTATGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGAATTCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTC SEQ ID NO: 24.NUCLEOTIDE SEQUENCE OF PLASMID 5259GGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCAAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGGTCGACAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACTCACCGTCCGGATCTCAGCAAGCAGGTATGTACTCTCCAGGGTGGGCCTGGCTTCCCCAGTCAAGACTCCAGGGATTTGAGGGACGCTGTGGGCTCTTCTCTTACATGTACCTTTTGCTTGCCTCAACCCTGACTATCTTCCAGGTCAGGATCCCAGAGTCAGGGGTCTGTATTTTCCTGCTGGTGGCTCCAGTTCAGGAACAGTAAACCCTGCTCCGAATATTGCCTCTCACATCTCGTCAATCTCCGCGAGGACTGGGGACCCTGTGACGAACATGGCTAGCAAGGCTGTGCTGCTTGCCCTGTTGATGGCAGGCTTGGCCCTGCAGCCAGGCACTGCCCTGCTGTGCTACTCCTGCAAAGCCCAGGTGAGCAACGAGGACTGCCTGCAGGTGGAGAACTGCACCCAGCTGGGGGAGCAGTGCTGGACCGCGCGCATCCGCGCAGTTGGCCTCCTGACCGTCATCAGCAAAGGCTGCAGCTTGAACTGCGTGGATGACTCACAGGACTACTACGTGGGCAAGAAGAACATCACGTGCTGTGACACCGACTTGTGCAACGCCAGCGGGGCCCATGCCCTGCAGCCGGCTGCCGCCATCCTTGCGCTGCTCCCTGCACTCGGCCTGCTGCTCTGGGGACCCGGCCAGCTATAGAGATCTGGGCCCTAACAAAACAAAAAGATGGGGTTATTCCCTAAACTTCATGGGTTACGTAATTGGAAGTTGGGGGACATTGCCACAAGATCATATTGTACAAAAGATCAAACACTGTTTTAGAAAACTTCCTGTAAACAGGCCTATTGATTGGAAAGTATGTCAAAGGATTGTGGGTCTTTTGGGCTTTGCTGCTCCATTTACACAATGTGGATATCCTGCCTTAATGCCTTTGTATGCATGTATACAAGCTAAACAGGCTTTCACTTTCTCGCCAACTTACAAGGCCTTTCTAAGTAAACAGTACATGAACCTTTACCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGGCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCCGGTCTGGAGCAAAGCTCATAGGAACTGACAATTCTGTCGTCCTCTCGCGGAAATATACATCGTTTCGATCTACGTATGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGAATTCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTC SEQ ID NO: 25.NUCLEOTIDE SEQUENCE OF PLASMID 5297GGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCAAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGGTCGACAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACTCACCGTCCGGATCTCAGCAAGCAGGTATGTACTCTCCAGGGTGGGCCTGGCTTCCCCAGTCAAGACTCCAGGGATTTGAGGGACGCTGTGGGCTCTTCTCTTACATGTACCTTTTGCTTGCCTCAACCCTGACTATCTTCCAGGTCAGGATCCCAGAGTCAGGGGTCTGTATTTTCCTGCTGGTGGCTCCAGTTCAGGAACAGTAAACCCTGCTCCGAATATTGCCTCTCACATCTCGTCAATCTCCGCGAGGACTGGGGACCCTGTGACGAACATGGCTAGCATTGTGGGAGGCTGGGAGTGCGAGAAGCATTCCCAACCCTGGCAGGTGCTTGTGGCCTCTCGTGGCAGGGCAGTCTGCGGCGGTGTTCTGGTGCACCCCCAGTGGGTCCTCACAGCTGCCCACTGCATCAGGAACAAAAGCGTGATCTTGCTGGGTCGGCACAGCTTGTTTCATCCTGAAGACACAGGCCAGGTATTTCAGGTCAGCCACAGCTTCCCACACCCGCTCTACGATATGAGCCTCCTGAAGAATCGATTCCTCAGGCCAGGTGATGACTCCAGCCACGACCTCATGCTGCTCCGCCTGTCAGAGCCTGCCGAGCTCACGGATGCTGTGAAGGTCATGGACCTGCCCACCCAGGAGCCAGCACTGGGGACCACCTGCTACGCCTCAGGCTGGGGCAGCATTGAACCAGAGGAGTTCTTGACCCCAAAGAAACTTCAGTGTGTGGACCTCCATGTTATTTCCAATGACGTGTGTGCGCAAGTTCACCCTCAGAAGGTGACCAAGTTCATGCTGTGTGCTGGACGCTGGACAGGGGGCAAAAGCACCTGCTCGGGTGATTCTGGGGGCCCACTTGTCTGTAATGGTGTGCTTCAAGGTATCACGTCATGGGGCAGTGAACCATGTGCCCTGCCCGAAAGGCCTTCCCTGTACACCAAGGTGGTGCATTACCGGAAGTGGATCAAGGACACCATCGTGGCCAACCCCTGAAGATCTGGGCCCTAACAAAACAAAAAGATGGGGTTATTCCCTAAACTTCATGGGTTACGTAATTGGAAGTTGGGGGACATTGCCACAAGATCATATTGTACAAAAGATCAAACACTGTTTTAGAAAACTTCCTGTAAACAGGCCTATTGATTGGAAAGTATGTCAAAGGATTGTGGGTCTTTTGGGCTTTGCTGCTCCATTTACACAATGTGGATATCCTGCCTTAATGCCTTTGTATGCATGTATACAAGCTAAACAGGCTTTCACTTTCTCGCCAACTTACAAGGCCTTTCTAAGTAAACAGTACATGAACCTTTACCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGGCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCCGGTCTGGAGCAAAGCTCATAGGAACTGACAATTCTGTCGTCCTCTCGCGGAAATATACATCGTTTCGATCTACGTATGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGAATTCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGT TGCCTGACTC SEQID NO: 26. NUCLEOTIDE SEQUENCE OF PLASMID 460GAATTCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCGGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCAAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGGGTACCAATCTTCCGAGTGAGAGACACAAAAAATTCCAACACACTATTGCAATGAAAATAAATTTCCTTTATTAGCCAGAAGTCAGATGCTCAAGGGGCTTCATGATGTCCCCATAATTTTTGGCAGAGGGAAAAAGATCATACGTAGATCGAAACGATGTATATTTCCGCGAGAGGACGACAGAATTGTCAGTTCCTATGAGCTTTGCTCCAGACCGGCTGCGAGCAAAACAAGCGGCTAGGAGTTCCGCAGTATGGATCGGCAGAGGAGCCACAAAGGTTCCACGCATGCGCTGATGGCCTATGGCCAAGCCCCAGCCAGTGGGGGTTGCGTCAGCAAACACTTGGCACAGACCAGGCCGTTGCCGAGCAACGGGGTAAAGGTTCATGTACTGTTTACTTAGAAAGGCCTTGTAAGTTGGCGAGAAAGTGAAAGCCTGTTTAGCTTGTATACATGCATACAAAGGCATTAAGGCAGGATATCCACATTGTGTAAATGGAGCAGCAAAGCCCAAAAGACCCACAATCCTTTGACATACTTTCCAATCAATAGGCCTGTTTACAGGAAGTTTTCTAAAACAGTGTTTGATCTTTTGTACAATATGATCTTGTGGCAATGTCCCCCAACTTCCAATTACGTAACCCATGAAGTTTAGGGAATAACCCCATCTTTTTGTTTTGTTAGGGCCCAGATCTTTAGGCTACTTCACTCAAAGTCTCTGCAGCTGCCTGCACTGTGAAGGCTGCAACATAAATCTGTCTCTTCACTTCTCCCCAGGCCTTGGAAGGGTCCACTTTGCTTTCAATATCAAACAGAGCATCATAAATTCCTGGGAATGACTCCCCTGCATACTTGTTGTGGCTGCTTGGAGCATAGATGACATGCCTATAAAAAGGCCTGTCTGGTAACCCTAATGGATCAATAAATGCTCTTTCCAGAAACATGAGTTGATCATTCATCATTCTTAATACTATTGGGTTGCTTTTGTCAAAGTCCTGGAGTCTCTCACTGAACTTGGAAGCAATTTCTGTAAAATTCTTTACTGCAGAAAAAAGTGAATCAAATGATACACTGTATGTCTTCATTTCCTGTGGATGTTTCATAGAAATACTGTAGATTTTGTCAGCATACTTTCTTAAAACTACAGCATAATCTCGACAATCAAAAGGGAGCACTATGGAATTGGCCAGCTCAAACACCATCCCTCCTCGAACCTGGGCCACAGTGAGGTGATATTTAAACATTGGATCATAAAACTTTTCCACCAACTCATATGTTTCATAGACACTGTGATACAGTGGATAGCCGCTGAATTTGTTTGTTTCCCAATTTTTAGTATACCGTGCTCTGCCTGAAGCAATTCCAAGTCGTTGGAAGAACACCTCAAAATCATTTCCAGATCCCAATTTGCTTATCCTGGGCATGCCACTGAACTCTGGGGAAGGACTTTTTTTAGTCCAACTTTCATAAAGAGATTTGCCTTCAAAGCCTTCATCAGGGCTTTTCAGCTCTTTTGTTAGGTTGTGTACCAAGCTGTACATCAGCGGTGTACAATCAACTCTCAGAGTGTAGTTTCCTTCTATAGATGAGTCAGCATTAATATAAGCCACGCCACGCTCTTGAAGGAGTCTTGAATTCTCCTCTGCCCACTCAGTAGAACCAAGAAGACCAAATTCTTCTGCATCCCAGCTTGCAAACAAAATTGTTCTTCTAGGTCTCCACCCTTCCTTTTTCAGTGTTCCAAAGCTCCTCACAATTTCATGAACAACAGCTGCTCCACTCTGAGGGTCAATACCACCAAACACCCATGAGTCCCGGTGACCTCCCAGAATGACATATCTGTCTGGTTCCACTGCTCCTCTGAGAGTACCTATCACATTGTAAATTCTTGTCACTTCATTGGTAGAGTGGATGTGCATCTTGACTTTTTGTGTAGAAAAGTTTCCAGTAAAGCCAGGTCCAACATTGTAGGGCACTTTGAGACTTCCTCTCCAGCTGCTATCTGGTGGTGCTGAGCCACCCATTTTTTCTAGGAGCTTCTGTGCATCATAGTATCCAATTGGATGAACAGGAATACTTGGAAGACCAACAGCCTCTGCAATTCCACGCCTATAAGCATATTCATTTGCTGGGTAACCTGGTGTGAGAGGGTCTCCTGCACCATTCAGATTTAGGATATTTCCACGCTGGACACCACCTCCAGGAAGATTCCAACCATCTGGATAGGACTTCACCCCAGGAGCAAAGTAGTCAGCAGGGTCGGAGTAGAGAATGACTCCTTTGGCCCCTGCCAGCTGGGCATTTTTAACCTTATTTCCTCTGAAAACTTTCCCATATCTGGCAATTACAATTTTCCCAGAGCAATTGATTTTCATGTCCCGTTCCAATTTAAAGAAGTCTTCAGTTCGTGCATAGTTAACATACACTAGATCGCCCTCTGGCATTCCTTGAGGAGAGAAAGCACTGAAAGGTGGTACAATATCCGAAACATTTTCATATCCTGGAGGAGGTGGTTCAAATAATGATGTGTTGAAAATCTCATTTCCATCTTCATTAATTATTGAGATGTAGTTGGGATGAGTCTTATTTGGGTAGGACAACAGGACATCATAATGTGCCAGCTCAACAGAATCCAGGCCAAATTCTTTCCACTGGGATTGAATTTGCTTTGCAAGCTGAAAGTTTTGTTCTGTTCCTGCTAAATGTGGTATCTGTGTAAAATTATATAAGAACTTCTTGATGTTCTCAGCTTTCAATTCATCCAAAAATGCTTTCATATTATGCTTTGGAGTAATGTTAGTAGCTTCATTGGAGGATTTTATAAACCACCCGAAGAGGAAGCCGAGGAGAAAGAAGCCACCCGCCAGCACCAGCGCCCCAGCGCACAGCCAGCGCGGGCGGCGCGCGCTAGCCATGTTCGTCACAGGGTCCCCAGTCCTCGCGGAGATTGACGAGATGTGAGAGGCAATATTCGGAGCAGGGTTTACTGTTCCTGAACTGGAGCCACCAGCAGGAAAATACAGACCCCTGACTCTGGGATCCTGACCTGGAAGATAGTCAGGGTTGAGGCAAGCAAAAGGTACATGTAAGAGAAGAGCCCACAGCGTCCCTCAAATCCCTGGAGTCTTGACTGGGGAAGCCAGGCCCACCCTGGAGAGTACATACCTGCTTGCTGAGATCCGGACGGTGAGTCACTCTTGGCACGGGGAATCCGCGTTCCAATGCACCGTTCCCGGCCGCGGAGGCTGGATCGGTCCCGGTGTCTTCTATGGAGGTCAAAACAGCGTGGATGGCGTCTCCAGGCGATCTGACGGTTCACTAAACGAGCTCTGCTTATATAGACCTCCCACCGTACACGCCTACCGCCCATTTGCGTCAACGGGGCGGGGTTATTACGACATTTTGGAAAGTCCCGTTGATTTTGGTGCTCGACCTGCAGGGTACCAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACTCACCGTCCGGATCTCAGCAAGCAGGTATGTACTCTCCAGGGTGGGCCTGGCTTCCCCAGTCAAGACTCCAGGGATTTGAGGGACGCTGTGGGCTCTTCTCTTACATGTACCTTTTGCTTGCCTCAACCCTGACTATCTTCCAGGTCAGGATCCCAGAGTCAGGGGTCTGTATTTTCCTGCTGGTGGCTCCAGTTCAGGAACAGTAAACCCTGCTCCGAATATTGCCTCTCACATCTCGTCAATCTCCGCGAGGACTGGGGACCCTGTGACGAACATGGCTAGCAAGGCTGTGCTGCTTGCCCTGTTGATGGCAGGCTTGGCCCTGCAGCCAGGCACTGCCCTGCTGTGCTACTCCTGCAAAGCCCAGGTGAGCAACGAGGACTGCCTGCAGGTGGAGAACTGCACCCAGCTGGGGGAGCAGTGCTGGACCGCGCGCATCCGCGCAGTTGGCCTCCTGACCGTCATCAGCAAAGGCTGCAGCTTGAACTGCGTGGATGACTCACAGGACTACTACGTGGGCAAGAAGAACATCACGTGCTGTGACACCGACTTGTGCAACGCCAGCGGGGCCCATGCCCTGCAGCCGGCTGCCGCCATCCTTGCGCTGCTCCCTGCACTCGGCCTGCTGCTCTGGGGACCCGGCCAGCTATAGAGATCTGGGCCCTAACAAAACAAAAAGATGGGGTTATTCCCTAAACTTCATGGGTTACGTAATTGGAAGTTGGGGGACATTGCCACAAGATCATATTGTACAAAAGATCAAACACTGTTTTAGAAAACTTCCTGTAAACAGGCCTATTGATTGGAAAGTATGTCAAAGGATTGTGGGTCTTTTGGGCTTTGCTGCTCCATTTACACAATGTGGATATCCTGCCTTAATGCCTTTGTATGCATGTATACAAGCTAAACAGGCTTTCACTTTCTCGCCAACTTACAAGGCCTTTCTAAGTAAACAGTACATGAACCTTTACCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGGCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCCGGTCTGGAGCAAAGCTCATAGGAACTGACAATTCTGTCGTCCTCTCGCGGAAATATACATCGTTTCGATCTACGTATGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGC SEQ ID NO: 27. NUCLEOTIDESEQUENCE OF PLASMID 451GGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCAAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGGTCGACAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACTCACCGTCCGGATCTCAGCAAGCAGGTATGTACTCTCCAGGGTGGGCCTGGCTTCCCCAGTCAAGACTCCAGGGATTTGAGGGACGCTGTGGGCTCTTCTCTTACATGTACCTTTTGCTTGCCTCAACCCTGACTATCTTCCAGGTCAGGATCCCAGAGTCAGGGGTCTGTATTTTCCTGCTGGTGGCTCCAGTTCAGGAACAGTAAACCCTGCTCCGAATATTGCCTCTCACATCTCGTCAATCTCCGCGAGGACTGGGGACCCTGTGACGAACATGGCTAGCGCGCGCCGCCCGCGCTGGCTGTGCGCTGGGGCGCTGGTGCTGGCGGGTGGCTTCTTTCTCCTCGGCTTCCTCTTCGGGTGGTTTATAAAATCCTCCAATGAAGCTACTAACATTACTCCAAAGCATAATATGAAAGCATTTTTGGATGAATTGAAAGCTGAGAACATCAAGAAGTTCTTATATAATTTTACACAGATACCACATTTAGCAGGAACAGAACAAAACTTTCAGCTTGCAAAGCAAATTCAATCCCAGTGGAAAGAATTTGGCCTGGATTCTGTTGAGCTGGCACATTATGATGTCCTGTTGTCCTACCCAAATAAGACTCATCCCAACTACATCTCAATAATTAATGAAGATGGAAATGAGATTTTCAACACATCATTATTTGAACCACCTCCTCCAGGATATGAAAATGTTTCGGATATTGTACCACCTTTCAGTGCTTTCTCTCCTCAAGGAATGCCAGAGGGCGATCTAGTGTATGTTAACTATGCACGAACTGAAGACTTCTTTAAATTGGAACGGGACATGAAAATCAATTGCTCTGGGAAAATTGTAATTGCCAGATATGGGAAAGTTTTCAGAGGAAATAAGGTTAAAAATGCCCAGCTGGCAGGGGCCAAAGGAGTCATTCTCTACTCCGACCCTGCTGACTACTTTGCTCCTGGGGTGAAGTCCTATCCAGATGGTTGGAATCTTCCTGGAGGTGGTGTCCAGCGTGGAAATATCCTAAATCTGAATGGTGCAGGAGACCCTCTCACACCAGGTTACCCAGCAAATGAATATGCTTATAGGCGTGGAATTGCAGAGGCTGTTGGTCTTCCAAGTATTCCTGTTCATCCAATTGGATACTATGATGCACAGAAGCTCCTAGAAAAAATGGGTGGCTCAGCACCACCAGATAGCAGCTGGAGAGGAAGTCTCAAAGTGCCCTACAATGTTGGACCTGGCTTTACTGGAAACTTTTCTACACAAAAAGTCAAGATGCACATCCACTCTACCAATGAAGTGACAAGAATTTACAATGTGATAGGTACTCTCAGAGGAGCAGTGGAACCAGACAGATATGTCATTCTGGGAGGTCACCGGGACTCATGGGTGTTTGGTGGTATTGACCCTCAGAGTGGAGCAGCTGTTGTTCATGAAATTGTGAGGAGCTTTGGAACACTGAAAAAGGAAGGGTGGAGACCTAGAAGAACAATTTTGTTTGCAAGCTGGGATGCAGAAGAATTTGGTCTTCTTGGTTCTACTGAGTGGGCAGAGGAGAATTCAAGACTCCTTCAAGAGCGTGGCGTGGCTTATATTAATGCTGACTCATCTATAGAAGGAAACTACACTCTGAGAGTTGATTGTACACCGCTGATGTACAGCTTGGTACACAACCTAACAAAAGAGCTGAAAAGCCCTGATGAAGGCTTTGAAGGCAAATCTCTTTATGAAAGTTGGACTAAAAAAAGTCCTTCCCCAGAGTTCAGTGGCATGCCCAGGATAAGCAAATTGGGATCTGGAAATGATTTTGAGGTGTTCTTCCAACGACTTGGAATTGCTTCAGGCAGAGCACGGTATACTAAAAATTGGGAAACAAACAAATTCAGCGGCTATCCACTGTATCACAGTGTCTATGAAACATATGAGTTGGTGGAAAAGTTTTATGATCCAATGTTTAAATATCACCTCACTGTGGCCCAGGTTCGAGGAGGGATGGTGTTTGAGCTGGCCAATTCCATAGTGCTCCCTTTTGATTGTCGAGATTATGCTGTAGTTTTAAGAAAGTATGCTGACAAAATCTACAGTATTTCTATGAAACATCCACAGGAAATGAAGACATACAGTGTATCATTTGATTCACTTTTTTCTGCAGTAAAGAATTTTACAGAAATTGCTTCCAAGTTCAGTGAGAGACTCCAGGACTTTGACAAAAGCAACCCAATAGTATTAAGAATGATGAATGATCAACTCATGTTTCTGGAAAGAGCATTTATTGATCCATTAGGGTTACCAGACAGGCCTTTTTATAGGCATGTCATCTATGCTCCAAGCAGCCACAACAAGTATGCAGGGGAGTCATTCCCAGGAATTTATGATGCTCTGTTTGATATTGAAAGCAAAGTGGACCCTTCCAAGGCCTGGGGAGAAGTGAAGAGACAGATTTATGTTGCAGCCTTCACAGTGCAGGCAGCTGCAGAGACTTTGAGTGAAGTAGCCGGATCCGAAGGTAGGGGTTCATTATTGACCTGTGGAGATGTCGAAGAAAACCCAGGACCCGCAAGCAAGGCTGTGCTGCTTGCCCTGTTGATGGCAGGCTTGGCCCTGCAGCCAGGCACTGCCCTGCTGTGCTACTCCTGCAAAGCCCAGGTGAGCAACGAGGACTGCCTGCAGGTGGAGAACTGCACCCAGCTGGGGGAGCAGTGCTGGACCGCGCGCATCCGCGCAGTTGGCCTCCTGACCGTCATCAGCAAAGGCTGCAGCTTGAACTGCGTGGATGACTCACAGGACTACTACGTGGGCAAGAAGAACATCACGTGCTGTGACACCGACTTGTGCAACGCCAGCGGGGCCCATGCCCTGCAGCCGGCTGCCGCCATCCTTGCGCTGCTCCCTGCACTCGGCCTGCTGCTCTGGGGACCCGGCCAGCTATAGAGATCTGGGCCCTAACAAAACAAAAAGATGGGGTTATTCCCTAAACTTCATGGGTTACGTAATTGGAAGTTGGGGGACATTGCCACAAGATCATATTGTACAAAAGATCAAACACTGTTTTAGAAAACTTCCTGTAAACAGGCCTATTGATTGGAAAGTATGTCAAAGGATTGTGGGTCTTTTGGGCTTTGCTGCTCCATTTACACAATGTGGATATCCTGCCTTAATGCCTTTGTATGCATGTATACAAGCTAAACAGGCTTTCACTTTCTCGCCAACTTACAAGGCCTTTCTAAGTAAACAGTACATGAACCTTTACCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGGCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCCGGTCTGGAGCAAAGCTCATAGGAACTGACAATTCTGTCGTCCTCTCGCGGAAATATACATCGTTTCGATCTACGTATGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGAATTCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTC SEQ ID NO: 28.NUCLEOTIDE SEQUENCE OF PLASMID 454GGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCAAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGGTCGACAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACTCACCGTCCGGATCTCAGCAAGCAGGTATGTACTCTCCAGGGTGGGCCTGGCTTCCCCAGTCAAGACTCCAGGGATTTGAGGGACGCTGTGGGCTCTTCTCTTACATGTACCTTTTGCTTGCCTCAACCCTGACTATCTTCCAGGTCAGGATCCCAGAGTCAGGGGTCTGTATTTTCCTGCTGGTGGCTCCAGTTCAGGAACAGTAAACCCTGCTCCGAATATTGCCTCTCACATCTCGTCAATCTCCGCGAGGACTGGGGACCCTGTGACGAACATGGCTAGCAAGGCTGTGCTGCTTGCCCTGTTGATGGCAGGCTTGGCCCTGCAGCCAGGCACTGCCCTGCTGTGCTACTCCTGCAAAGCCCAGGTGAGCAACGAGGACTGCCTGCAGGTGGAGAACTGCACCCAGCTGGGGGAGCAGTGCTGGACCGCGCGCATCCGCGCAGTTGGCCTCCTGACCGTCATCAGCAAAGGCTGCAGCTTGAACTGCGTGGATGACTCACAGGACTACTACGTGGGCAAGAAGAACATCACGTGCTGTGACACCGACTTGTGCAACGCCAGCGGGGCCCATGCCCTGCAGCCGGCTGCCGCCATCCTTGCGCTGCTCCCTGCACTCGGCCTGCTGCTCTGGGGACCCGGCCAGCTAGGATCCCAGACCCTGAACTTTGATCTGCTGAAACTGGCAGGCGATGTGGAAAGCAACCCAGGCCCAATGGCAAGCGCGCGCCGCCCGCGCTGGCTGTGCGCTGGGGCGCTGGTGCTGGCGGGTGGCTTCTTTCTCCTCGGCTTCCTCTTCGGGTGGTTTATAAAATCCTCCAATGAAGCTACTAACATTACTCCAAAGCATAATATGAAAGCATTTTTGGATGAATTGAAAGCTGAGAACATCAAGAAGTTCTTATATAATTTTACACAGATACCACATTTAGCAGGAACAGAACAAAACTTTCAGCTTGCAAAGCAAATTCAATCCCAGTGGAAAGAATTTGGCCTGGATTCTGTTGAGCTGGCACATTATGATGTCCTGTTGTCCTACCCAAATAAGACTCATCCCAACTACATCTCAATAATTAATGAAGATGGAAATGAGATTTTCAACACATCATTATTTGAACCACCTCCTCCAGGATATGAAAATGTTTCGGATATTGTACCACCTTTCAGTGCTTTCTCTCCTCAAGGAATGCCAGAGGGCGATCTAGTGTATGTTAACTATGCACGAACTGAAGACTTCTTTAAATTGGAACGGGACATGAAAATCAATTGCTCTGGGAAAATTGTAATTGCCAGATATGGGAAAGTTTTCAGAGGAAATAAGGTTAAAAATGCCCAGCTGGCAGGGGCCAAAGGAGTCATTCTCTACTCCGACCCTGCTGACTACTTTGCTCCTGGGGTGAAGTCCTATCCAGATGGTTGGAATCTTCCTGGAGGTGGTGTCCAGCGTGGAAATATCCTAAATCTGAATGGTGCAGGAGACCCTCTCACACCAGGTTACCCAGCAAATGAATATGCTTATAGGCGTGGAATTGCAGAGGCTGTTGGTCTTCCAAGTATTCCTGTTCATCCAATTGGATACTATGATGCACAGAAGCTCCTAGAAAAAATGGGTGGCTCAGCACCACCAGATAGCAGCTGGAGAGGAAGTCTCAAAGTGCCCTACAATGTTGGACCTGGCTTTACTGGAAACTTTTCTACACAAAAAGTCAAGATGCACATCCACTCTACCAATGAAGTGACAAGAATTTACAATGTGATAGGTACTCTCAGAGGAGCAGTGGAACCAGACAGATATGTCATTCTGGGAGGTCACCGGGACTCATGGGTGTTTGGTGGTATTGACCCTCAGAGTGGAGCAGCTGTTGTTCATGAAATTGTGAGGAGCTTTGGAACACTGAAAAAGGAAGGGTGGAGACCTAGAAGAACAATTTTGTTTGCAAGCTGGGATGCAGAAGAATTTGGTCTTCTTGGTTCTACTGAGTGGGCAGAGGAGAATTCAAGACTCCTTCAAGAGCGTGGCGTGGCTTATATTAATGCTGACTCATCTATAGAAGGAAACTACACTCTGAGAGTTGATTGTACACCGCTGATGTACAGCTTGGTACACAACCTAACAAAAGAGCTGAAAAGCCCTGATGAAGGCTTTGAAGGCAAATCTCTTTATGAAAGTTGGACTAAAAAAAGTCCTTCCCCAGAGTTCAGTGGCATGCCCAGGATAAGCAAATTGGGATCTGGAAATGATTTTGAGGTGTTCTTCCAACGACTTGGAATTGCTTCAGGCAGAGCACGGTATACTAAAAATTGGGAAACAAACAAATTCAGCGGCTATCCACTGTATCACAGTGTCTATGAAACATATGAGTTGGTGGAAAAGTTTTATGATCCAATGTTTAAATATCACCTCACTGTGGCCCAGGTTCGAGGAGGGATGGTGTTTGAGCTGGCCAATTCCATAGTGCTCCCTTTTGATTGTCGAGATTATGCTGTAGTTTTAAGAAAGTATGCTGACAAAATCTACAGTATTTCTATGAAACATCCACAGGAAATGAAGACATACAGTGTATCATTTGATTCACTTTTTTCTGCAGTAAAGAATTTTACAGAAATTGCTTCCAAGTTCAGTGAGAGACTCCAGGACTTTGACAAAAGCAACCCAATAGTATTAAGAATGATGAATGATCAACTCATGTTTCTGGAAAGAGCATTTATTGATCCATTAGGGTTACCAGACAGGCCTTTTTATAGGCATGTCATCTATGCTCCAAGCAGCCACAACAAGTATGCAGGGGAGTCATTCCCAGGAATTTATGATGCTCTGTTTGATATTGAAAGCAAAGTGGACCCTTCCAAGGCCTGGGGAGAAGTGAAGAGACAGATTTATGTTGCAGCCTTCACAGTGCAGGCAGCTGCAGAGACTTTGAGTGAAGTAGCCTAAAGATCTGGGCCCTAACAAAACAAAAAGATGGGGTTATTCCCTAAACTTCATGGGTTACGTAATTGGAAGTTGGGGGACATTGCCACAAGATCATATTGTACAAAAGATCAAACACTGTTTTAGAAAACTTCCTGTAAACAGGCCTATTGATTGGAAAGTATGTCAAAGGATTGTGGGTCTTTTGGGCTTTGCTGCTCCATTTACACAATGTGGATATCCTGCCTTAATGCCTTTGTATGCATGTATACAAGCTAAACAGGCTTTCACTTTCTCGCCAACTTACAAGGCCTTTCTAAGTAAACAGTACATGAACCTTTACCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGGCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCCGGTCTGGAGCAAAGCTCATAGGAACTGACAATTCTGTCGTCCTCTCGCGGAAATATACATCGTTTCGATCTACGTATGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGAATTCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTC SEQ ID NO: 29.NUCLEOTIDE SEQUENCE OF PLASMID 5300GGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCAAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGGTCGACAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACTCACCGTCCGGATCTCAGCAAGCAGGTATGTACTCTCCAGGGTGGGCCTGGCTTCCCCAGTCAAGACTCCAGGGATTTGAGGGACGCTGTGGGCTCTTCTCTTACATGTACCTTTTGCTTGCCTCAACCCTGACTATCTTCCAGGTCAGGATCCCAGAGTCAGGGGTCTGTATTTTCCTGCTGGTGGCTCCAGTTCAGGAACAGTAAACCCTGCTCCGAATATTGCCTCTCACATCTCGTCAATCTCCGCGAGGACTGGGGACCCTGTGACGAACATGGCTAGCATTGTGGGAGGCTGGGAGTGCGAGAAGCATTCCCAACCCTGGCAGGTGCTTGTGGCCTCTCGTGGCAGGGCAGTCTGCGGCGGTGTTCTGGTGCACCCCCAGTGGGTCCTCACAGCTGCCCACTGCATCAGGAACAAAAGCGTGATCTTGCTGGGTCGGCACAGCTTGTTTCATCCTGAAGACACAGGCCAGGTATTTCAGGTCAGCCACAGCTTCCCACACCCGCTCTACGATATGAGCCTCCTGAAGAATCGATTCCTCAGGCCAGGTGATGACTCCAGCCACGACCTCATGCTGCTCCGCCTGTCAGAGCCTGCCGAGCTCACGGATGCTGTGAAGGTCATGGACCTGCCCACCCAGGAGCCAGCACTGGGGACCACCTGCTACGCCTCAGGCTGGGGCAGCATTGAACCAGAGGAGTTCTTGACCCCAAAGAAACTTCAGTGTGTGGACCTCCATGTTATTTCCAATGACGTGTGTGCGCAAGTTCACCCTCAGAAGGTGACCAAGTTCATGCTGTGTGCTGGACGCTGGACAGGGGGCAAAAGCACCTGCTCGGGTGATTCTGGGGGCCCACTTGTCTGTAATGGTGTGCTTCAAGGTATCACGTCATGGGGCAGTGAACCATGTGCCCTGCCCGAAAGGCCTTCCCTGTACACCAAGGTGGTGCATTACCGGAAGTGGATCAAGGACACCATCGTGGCCAACCCCGGATCCCAGACCCTGAACTTTGATCTGCTGAAACTGGCAGGCGATGTGGAAAGCAACCCAGGCCCAATGGCAAGCGCGCGCCGCCCGCGCTGGCTGTGCGCTGGGGCGCTGGTGCTGGCGGGTGGCTTCTTTCTCCTCGGCTTCCTCTTCGGGTGGTTTATAAAATCCTCCAATGAAGCTACTAACATTACTCCAAAGCATAATATGAAAGCATTTTTGGATGAATTGAAAGCTGAGAACATCAAGAAGTTCTTATATAATTTTACACAGATACCACATTTAGCAGGAACAGAACAAAACTTTCAGCTTGCAAAGCAAATTCAATCCCAGTGGAAAGAATTTGGCCTGGATTCTGTTGAGCTGGCACATTATGATGTCCTGTTGTCCTACCCAAATAAGACTCATCCCAACTACATCTCAATAATTAATGAAGATGGAAATGAGATTTTCAACACATCATTATTTGAACCACCTCCTCCAGGATATGAAAATGTTTCGGATATTGTACCACCTTTCAGTGCTTTCTCTCCTCAAGGAATGCCAGAGGGCGATCTAGTGTATGTTAACTATGCACGAACTGAAGACTTCTTTAAATTGGAACGGGACATGAAAATCAATTGCTCTGGGAAAATTGTAATTGCCAGATATGGGAAAGTTTTCAGAGGAAATAAGGTTAAAAATGCCCAGCTGGCAGGGGCCAAAGGAGTCATTCTCTACTCCGACCCTGCTGACTACTTTGCTCCTGGGGTGAAGTCCTATCCAGATGGTTGGAATCTTCCTGGAGGTGGTGTCCAGCGTGGAAATATCCTAAATCTGAATGGTGCAGGAGACCCTCTCACACCAGGTTACCCAGCAAATGAATATGCTTATAGGCGTGGAATTGCAGAGGCTGTTGGTCTTCCAAGTATTCCTGTTCATCCAATTGGATACTATGATGCACAGAAGCTCCTAGAAAAAATGGGTGGCTCAGCACCACCAGATAGCAGCTGGAGAGGAAGTCTCAAAGTGCCCTACAATGTTGGACCTGGCTTTACTGGAAACTTTTCTACACAAAAAGTCAAGATGCACATCCACTCTACCAATGAAGTGACAAGAATTTACAATGTGATAGGTACTCTCAGAGGAGCAGTGGAACCAGACAGATATGTCATTCTGGGAGGTCACCGGGACTCATGGGTGTTTGGTGGTATTGACCCTCAGAGTGGAGCAGCTGTTGTTCATGAAATTGTGAGGAGCTTTGGAACACTGAAAAAGGAAGGGTGGAGACCTAGAAGAACAATTTTGTTTGCAAGCTGGGATGCAGAAGAATTTGGTCTTCTTGGTTCTACTGAGTGGGCAGAGGAGAATTCAAGACTCCTTCAAGAGCGTGGCGTGGCTTATATTAATGCTGACTCATCTATAGAAGGAAACTACACTCTGAGAGTTGATTGTACACCGCTGATGTACAGCTTGGTACACAACCTAACAAAAGAGCTGAAAAGCCCTGATGAAGGCTTTGAAGGCAAATCTCTTTATGAAAGTTGGACTAAAAAAAGTCCTTCCCCAGAGTTCAGTGGCATGCCCAGGATAAGCAAATTGGGATCTGGAAATGATTTTGAGGTGTTCTTCCAACGACTTGGAATTGCTTCAGGCAGAGCACGGTATACTAAAAATTGGGAAACAAACAAATTCAGCGGCTATCCACTGTATCACAGTGTCTATGAAACATATGAGTTGGTGGAAAAGTTTTATGATCCAATGTTTAAATATCACCTCACTGTGGCCCAGGTTCGAGGAGGGATGGTGTTTGAGCTGGCCAATTCCATAGTGCTCCCTTTTGATTGTCGAGATTATGCTGTAGTTTTAAGAAAGTATGCTGACAAAATCTACAGTATTTCTATGAAACATCCACAGGAAATGAAGACATACAGTGTATCATTTGATTCACTTTTTTCTGCAGTAAAGAATTTTACAGAAATTGCTTCCAAGTTCAGTGAGAGACTCCAGGACTTTGACAAAAGCAACCCAATAGTATTAAGAATGATGAATGATCAACTCATGTTTCTGGAAAGAGCATTTATTGATCCATTAGGGTTACCAGACAGGCCTTTTTATAGGCATGTCATCTATGCTCCAAGCAGCCACAACAAGTATGCAGGGGAGTCATTCCCAGGAATTTATGATGCTCTGTTTGATATTGAAAGCAAAGTGGACCCTTCCAAGGCCTGGGGAGAAGTGAAGAGACAGATTTATGTTGCAGCCTTCACAGTGCAGGCAGCTGCAGAGACTTTGAGTGAAGTAGCCTAAAGATCTGGGCCCTAACAAAACAAAAAGATGGGGTTATTCCCTAAACTTCATGGGTTACGTAATTGGAAGTTGGGGGACATTGCCACAAGATCATATTGTACAAAAGATCAAACACTGTTTTAGAAAACTTCCTGTAAACAGGCCTATTGATTGGAAAGTATGTCAAAGGATTGTGGGTCTTTTGGGCTTTGCTGCTCCATTTACACAATGTGGATATCCTGCCTTAATGCCTTTGTATGCATGTATACAAGCTAAACAGGCTTTCACTTTCTCGCCAACTTACAAGGCCTTTCTAAGTAAACAGTACATGAACCTTTACCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGGCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCCGGTCTGGAGCAAAGCTCATAGGAACTGACAATTCTGTCGTCCTCTCGCGGAAATATACATCGTTTCGATCTACGTATGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGAATTCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTC SEQ ID NO: 30. NUCLEOTIDE SEQUENCE OF PLASMID 449GGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCAAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGGTCGACAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACTCACCGTCCGGATCTCAGCAAGCAGGTATGTACTCTCCAGGGTGGGCCTGGCTTCCCCAGTCAAGACTCCAGGGATTTGAGGGACGCTGTGGGCTCTTCTCTTACATGTACCTTTTGCTTGCCTCAACCCTGACTATCTTCCAGGTCAGGATCCCAGAGTCAGGGGTCTGTATTTTCCTGCTGGTGGCTCCAGTTCAGGAACAGTAAACCCTGCTCCGAATATTGCCTCTCACATCTCGTCAATCTCCGCGAGGACTGGGGACCCTGTGACGAACATGGCTAGCGCGCGCCGCCCGCGCTGGCTGTGCGCTGGGGCGCTGGTGCTGGCGGGTGGCTTCTTTCTCCTCGGCTTCCTCTTCGGGTGGTTTATAAAATCCTCCAATGAAGCTACTAACATTACTCCAAAGCATAATATGAAAGCATTTTTGGATGAATTGAAAGCTGAGAACATCAAGAAGTTCTTATATAATTTTACACAGATACCACATTTAGCAGGAACAGAACAAAACTTTCAGCTTGCAAAGCAAATTCAATCCCAGTGGAAAGAATTTGGCCTGGATTCTGTTGAGCTGGCACATTATGATGTCCTGTTGTCCTACCCAAATAAGACTCATCCCAACTACATCTCAATAATTAATGAAGATGGAAATGAGATTTTCAACACATCATTATTTGAACCACCTCCTCCAGGATATGAAAATGTTTCGGATATTGTACCACCTTTCAGTGCTTTCTCTCCTCAAGGAATGCCAGAGGGCGATCTAGTGTATGTTAACTATGCACGAACTGAAGACTTCTTTAAATTGGAACGGGACATGAAAATCAATTGCTCTGGGAAAATTGTAATTGCCAGATATGGGAAAGTTTTCAGAGGAAATAAGGTTAAAAATGCCCAGCTGGCAGGGGCCAAAGGAGTCATTCTCTACTCCGACCCTGCTGACTACTTTGCTCCTGGGGTGAAGTCCTATCCAGATGGTTGGAATCTTCCTGGAGGTGGTGTCCAGCGTGGAAATATCCTAAATCTGAATGGTGCAGGAGACCCTCTCACACCAGGTTACCCAGCAAATGAATATGCTTATAGGCGTGGAATTGCAGAGGCTGTTGGTCTTCCAAGTATTCCTGTTCATCCAATTGGATACTATGATGCACAGAAGCTCCTAGAAAAAATGGGTGGCTCAGCACCACCAGATAGCAGCTGGAGAGGAAGTCTCAAAGTGCCCTACAATGTTGGACCTGGCTTTACTGGAAACTTTTCTACACAAAAAGTCAAGATGCACATCCACTCTACCAATGAAGTGACAAGAATTTACAATGTGATAGGTACTCTCAGAGGAGCAGTGGAACCAGACAGATATGTCATTCTGGGAGGTCACCGGGACTCATGGGTGTTTGGTGGTATTGACCCTCAGAGTGGAGCAGCTGTTGTTCATGAAATTGTGAGGAGCTTTGGAACACTGAAAAAGGAAGGGTGGAGACCTAGAAGAACAATTTTGTTTGCAAGCTGGGATGCAGAAGAATTTGGTCTTCTTGGTTCTACTGAGTGGGCAGAGGAGAATTCAAGACTCCTTCAAGAGCGTGGCGTGGCTTATATTAATGCTGACTCATCTATAGAAGGAAACTACACTCTGAGAGTTGATTGTACACCGCTGATGTACAGCTTGGTACACAACCTAACAAAAGAGCTGAAAAGCCCTGATGAAGGCTTTGAAGGCAAATCTCTTTATGAAAGTTGGACTAAAAAAAGTCCTTCCCCAGAGTTCAGTGGCATGCCCAGGATAAGCAAATTGGGATCTGGAAATGATTTTGAGGTGTTCTTCCAACGACTTGGAATTGCTTCAGGCAGAGCACGGTATACTAAAAATTGGGAAACAAACAAATTCAGCGGCTATCCACTGTATCACAGTGTCTATGAAACATATGAGTTGGTGGAAAAGTTTTATGATCCAATGTTTAAATATCACCTCACTGTGGCCCAGGTTCGAGGAGGGATGGTGTTTGAGCTGGCCAATTCCATAGTGCTCCCTTTTGATTGTCGAGATTATGCTGTAGTTTTAAGAAAGTATGCTGACAAAATCTACAGTATTTCTATGAAACATCCACAGGAAATGAAGACATACAGTGTATCATTTGATTCACTTTTTTCTGCAGTAAAGAATTTTACAGAAATTGCTTCCAAGTTCAGTGAGAGACTCCAGGACTTTGACAAAAGCAACCCAATAGTATTAAGAATGATGAATGATCAACTCATGTTTCTGGAAAGAGCATTTATTGATCCATTAGGGTTACCAGACAGGCCTTTTTATAGGCATGTCATCTATGCTCCAAGCAGCCACAACAAGTATGCAGGGGAGTCATTCCCAGGAATTTATGATGCTCTGTTTGATATTGAAAGCAAAGTGGACCCTTCCAAGGCCTGGGGAGAAGTGAAGAGACAGATTTATGTTGCAGCCTTCACAGTGCAGGCAGCTGCAGAGACTTTGAGTGAAGTAGCCTAAAGATCTGACCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCAGCAAGGCTGTGCTGCTTGCCCTGTTGATGGCAGGCTTGGCCCTGCAGCCAGGCACTGCCCTGCTGTGCTACTCCTGCAAAGCCCAGGTGAGCAACGAGGACTGCCTGCAGGTGGAGAACTGCACCCAGCTGGGGGAGCAGTGCTGGACCGCGCGCATCCGCGCAGTTGGCCTCCTGACCGTCATCAGCAAAGGCTGCAGCTTGAACTGCGTGGATGACTCACAGGACTACTACGTGGGCAAGAAGAACATCACGTGCTGTGACACCGACTTGTGCAACGCCAGCGGGGCCCATGCCCTGCAGCCGGCTGCCGCCATCCTTGCGCTGCTCCCTGCACTCGGCCTGCTGCTCTGGGGACCCGGCCAGCTATAGGGATCTGGGCCCTAACAAAACAAAAAGATGGGGTTATTCCCTAAACTTCATGGGTTACGTAATTGGAAGTTGGGGGACATTGCCACAAGATCATATTGTACAAAAGATCAAACACTGTTTTAGAAAACTTCCTGTAAACAGGCCTATTGATTGGAAAGTATGTCAAAGGATTGTGGGTCTTTTGGGCTTTGCTGCTCCATTTACACAATGTGGATATCCTGCCTTAATGCCTTTGTATGCATGTATACAAGCTAAACAGGCTTTCACTTTCTCGCCAACTTACAAGGCCTTTCTAAGTAAACAGTACATGAACCTTTACCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGGCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCCGGTCTGGAGCAAAGCTCATAGGAACTGACAATTCTGTCGTCCTCTCGCGGAAATATACATCGTTTCGATCTACGTATGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGAATTCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCAT AGTTGCCTGACTCSEQ ID NO: 31. NUCLEOTIDE SEQUENCE OF PLASMID 603GGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCAAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGGTCGACAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACTCACCGTCCGGATCTCAGCAAGCAGGTATGTACTCTCCAGGGTGGGCCTGGCTTCCCCAGTCAAGACTCCAGGGATTTGAGGGACGCTGTGGGCTCTTCTCTTACATGTACCTTTTGCTTGCCTCAACCCTGACTATCTTCCAGGTCAGGATCCCAGAGTCAGGGGTCTGTATTTTCCTGCTGGTGGCTCCAGTTCAGGAACAGTAAACCCTGCTCCGAATATTGCCTCTCACATCTCGTCAATCTCCGCGAGGACTGGGGACCCTGTGACGAACATGGCTAGCAAGGCTGTGCTGCTTGCCCTGTTGATGGCAGGCTTGGCCCTGCAGCCAGGCACTGCCCTGCTGTGCTACTCCTGCAAAGCCCAGGTGAGCAACGAGGACTGCCTGCAGGTGGAGAACTGCACCCAGCTGGGGGAGCAGTGCTGGACCGCGCGCATCCGCGCAGTTGGCCTCCTGACCGTCATCAGCAAAGGCTGCAGCTTGAACTGCGTGGATGACTCACAGGACTACTACGTGGGCAAGAAGAACATCACGTGCTGTGACACCGACTTGTGCAACGCCAGCGGGGCCCATGCCCTGCAGCCGGCTGCCGCCATCCTTGCGCTGCTCCCTGCACTCGGCCTGCTGCTCTGGGGACCCGGCCAGCTATAGAGATCTGACCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACCATGGCGCGCCGCCCGCGCTGGCTGTGCGCTGGGGCGCTGGTGCTGGCGGGTGGCTTCTTTCTCCTCGGCTTCCTCTTCGGGTGGTTTATAAAATCCTCCAATGAAGCTACTAACATTACTCCAAAGCATAATATGAAAGCATTTTTGGATGAATTGAAAGCTGAGAACATCAAGAAGTTCTTATATAATTTTACACAGATACCACATTTAGCAGGAACAGAACAAAACTTTCAGCTTGCAAAGCAAATTCAATCCCAGTGGAAAGAATTTGGCCTGGATTCTGTTGAGCTGGCACATTATGATGTCCTGTTGTCCTACCCAAATAAGACTCATCCCAACTACATCTCAATAATTAATGAAGATGGAAATGAGATTTTCAACACATCATTATTTGAACCACCTCCTCCAGGATATGAAAATGTTTCGGATATTGTACCACCTTTCAGTGCTTTCTCTCCTCAAGGAATGCCAGAGGGCGATCTAGTGTATGTTAACTATGCACGAACTGAAGACTTCTTTAAATTGGAACGGGACATGAAAATCAATTGCTCTGGGAAAATTGTAATTGCCAGATATGGGAAAGTTTTCAGAGGAAATAAGGTTAAAAATGCCCAGCTGGCAGGGGCCAAAGGAGTCATTCTCTACTCCGACCCTGCTGACTACTTTGCTCCTGGGGTGAAGTCCTATCCAGATGGTTGGAATCTTCCTGGAGGTGGTGTCCAGCGTGGAAATATCCTAAATCTGAATGGTGCAGGAGACCCTCTCACACCAGGTTACCCAGCAAATGAATATGCTTATAGGCGTGGAATTGCAGAGGCTGTTGGTCTTCCAAGTATTCCTGTTCATCCAATTGGATACTATGATGCACAGAAGCTCCTAGAAAAAATGGGTGGCTCAGCACCACCAGATAGCAGCTGGAGAGGAAGTCTCAAAGTGCCCTACAATGTTGGACCTGGCTTTACTGGAAACTTTTCTACACAAAAAGTCAAGATGCACATCCACTCTACCAATGAAGTGACAAGAATTTACAATGTGATAGGTACTCTCAGAGGAGCAGTGGAACCAGACAGATATGTCATTCTGGGAGGTCACCGGGACTCATGGGTGTTTGGTGGTATTGACCCTCAGAGTGGAGCAGCTGTTGTTCATGAAATTGTGAGGAGCTTTGGAACACTGAAAAAGGAAGGGTGGAGACCTAGAAGAACAATTTTGTTTGCAAGCTGGGATGCAGAAGAATTTGGTCTTCTTGGTTCTACTGAGTGGGCAGAGGAGAATTCAAGACTCCTTCAAGAGCGTGGCGTGGCTTATATTAATGCTGACTCATCTATAGAAGGAAACTACACTCTGAGAGTTGATTGTACACCGCTGATGTACAGCTTGGTACACAACCTAACAAAAGAGCTGAAAAGCCCTGATGAAGGCTTTGAAGGCAAATCTCTTTATGAAAGTTGGACTAAAAAAAGTCCTTCCCCAGAGTTCAGTGGCATGCCCAGGATAAGCAAATTGGGATCTGGAAATGATTTTGAGGTGTTCTTCCAACGACTTGGAATTGCTTCAGGCAGAGCACGGTATACTAAAAATTGGGAAACAAACAAATTCAGCGGCTATCCACTGTATCACAGTGTCTATGAAACATATGAGTTGGTGGAAAAGTTTTATGATCCAATGTTTAAATATCACCTCACTGTGGCCCAGGTTCGAGGAGGGATGGTGTTTGAGCTGGCCAATTCCATAGTGCTCCCTTTTGATTGTCGAGATTATGCTGTAGTTTTAAGAAAGTATGCTGACAAAATCTACAGTATTTCTATGAAACATCCACAGGAAATGAAGACATACAGTGTATCATTTGATTCACTTTTTTCTGCAGTAAAGAATTTTACAGAAATTGCTTCCAAGTTCAGTGAGAGACTCCAGGACTTTGACAAAAGCAACCCAATAGTATTAAGAATGATGAATGATCAACTCATGTTTCTGGAAAGAGCATTTATTGATCCATTAGGGTTACCAGACAGGCCTTTTTATAGGCATGTCATCTATGCTCCAAGCAGCCACAACAAGTATGCAGGGGAGTCATTCCCAGGAATTTATGATGCTCTGTTTGATATTGAAAGCAAAGTGGACCCTTCCAAGGCCTGGGGAGAAGTGAAGAGACAGATTTATGTTGCAGCCTTCACAGTGCAGGCAGCTGCAGAGACTTTGAGTGAAGTAGCCTAAAGATCTGGGCCCTAACAAAACAAAAAGATGGGGTTATTCCCTAAACTTCATGGGTTACGTAATTGGAAGTTGGGGGACATTGCCACAAGATCATATTGTACAAAAGATCAAACACTGTTTTAGAAAACTTCCTGTAAACAGGCCTATTGATTGGAAAGTATGTCAAAGGATTGTGGGTCTTTTGGGCTTTGCTGCTCCATTTACACAATGTGGATATCCTGCCTTAATGCCTTTGTATGCATGTATACAAGCTAAACAGGCTTTCACTTTCTCGCCAACTTACAAGGCCTTTCTAAGTAAACAGTACATGAACCTTTACCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGGCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCCGGTCTGGAGCAAAGCTCATAGGAACTGACAATTCTGTCGTCCTCTCGCGGAAATATACATCGTTTCGATCTACGTATGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGAATTCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTC SEQ ID NO: 32. NUCLEOTIDE SEQUENCE OF PLASMID 455GGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCAAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGGTCGACAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACTCACCGTCCGGATCTCAGCAAGCAGGTATGTACTCTCCAGGGTGGGCCTGGCTTCCCCAGTCAAGACTCCAGGGATTTGAGGGACGCTGTGGGCTCTTCTCTTACATGTACCTTTTGCTTGCCTCAACCCTGACTATCTTCCAGGTCAGGATCCCAGAGTCAGGGGTCTGTATTTTCCTGCTGGTGGCTCCAGTTCAGGAACAGTAAACCCTGCTCCGAATATTGCCTCTCACATCTCGTCAATCTCCGCGAGGACTGGGGACCCTGTGACGAACATGGCTAGCAAGGCTGTGCTGCTTGCCCTGTTGATGGCAGGCTTGGCCCTGCAGCCAGGCACTGCCCTGCTGTGCTACTCCTGCAAAGCCCAGGTGAGCAACGAGGACTGCCTGCAGGTGGAGAACTGCACCCAGCTGGGGGAGCAGTGCTGGACCGCGCGCATCCGCGCAGTTGGCCTCCTGACCGTCATCAGCAAAGGCTGCAGCTTGAACTGCGTGGATGACTCACAGGACTACTACGTGGGCAAGAAGAACATCACGTGCTGTGACACCGACTTGTGCAACGCCAGCGGGGCCCATGCCCTGCAGCCGGCTGCCGCCATCCTTGCGCTGCTCCCTGCACTCGGCCTGCTGCTCTGGGGACCCGGCCAGCTATAGAGATCTGACCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCAGCATTGTGGGAGGCTGGGAGTGCGAGAAGCATTCCCAACCCTGGCAGGTGCTTGTGGCCTCTCGTGGCAGGGCAGTCTGCGGCGGTGTTCTGGTGCACCCCCAGTGGGTCCTCACAGCTGCCCACTGCATCAGGAACAAAAGCGTGATCTTGCTGGGTCGGCACAGCTTGTTTCATCCTGAAGACACAGGCCAGGTATTTCAGGTCAGCCACAGCTTCCCACACCCGCTCTACGATATGAGCCTCCTGAAGAATCGATTCCTCAGGCCAGGTGATGACTCCAGCCACGACCTCATGCTGCTCCGCCTGTCAGAGCCTGCCGAGCTCACGGATGCTGTGAAGGTCATGGACCTGCCCACCCAGGAGCCAGCACTGGGGACCACCTGCTACGCCTCAGGCTGGGGCAGCATTGAACCAGAGGAGTTCTTGACCCCAAAGAAACTTCAGTGTGTGGACCTCCATGTTATTTCCAATGACGTGTGTGCGCAAGTTCACCCTCAGAAGGTGACCAAGTTCATGCTGTGTGCTGGACGCTGGACAGGGGGCAAAAGCACCTGCTCGGGTGATTCTGGGGGCCCACTTGTCTGTAATGGTGTGCTTCAAGGTATCACGTCATGGGGCAGTGAACCATGTGCCCTGCCCGAAAGGCCTTCCCTGTACACCAAGGTGGTGCATTACCGGAAGTGGATCAAGGACACCATCGTGGCCAACCCCTGAGGATCTGGGCCCTAACAAAACAAAAAGATGGGGTTATTCCCTAAACTTCATGGGTTACGTAATTGGAAGTTGGGGGACATTGCCACAAGATCATATTGTACAAAAGATCAAACACTGTTTTAGAAAACTTCCTGTAAACAGGCCTATTGATTGGAAAGTATGTCAAAGGATTGTGGGTCTTTTGGGCTTTGCTGCTCCATTTACACAATGTGGATATCCTGCCTTAATGCCTTTGTATGCATGTATACAAGCTAAACAGGCTTTCACTTTCTCGCCAACTTACAAGGCCTTTCTAAGTAAACAGTACATGAACCTTTACCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGGCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCCGGTCTGGAGCAAAGCTCATAGGAACTGACAATTCTGTCGTCCTCTCGCGGAAATATACATCGTTTCGATCTACGTATGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGAATTCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTC SEQ ID NO: 33. NUCLEOTIDE SEQUENCE OFPLASMID 456 GGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCAAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGGTCGACAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACTCACCGTCCGGATCTCAGCAAGCAGGTATGTACTCTCCAGGGTGGGCCTGGCTTCCCCAGTCAAGACTCCAGGGATTTGAGGGACGCTGTGGGCTCTTCTCTTACATGTACCTTTTGCTTGCCTCAACCCTGACTATCTTCCAGGTCAGGATCCCAGAGTCAGGGGTCTGTATTTTCCTGCTGGTGGCTCCAGTTCAGGAACAGTAAACCCTGCTCCGAATATTGCCTCTCACATCTCGTCAATCTCCGCGAGGACTGGGGACCCTGTGACGAACATGGCTAGCATTGTGGGAGGCTGGGAGTGCGAGAAGCATTCCCAACCCTGGCAGGTGCTTGTGGCCTCTCGTGGCAGGGCAGTCTGCGGCGGTGTTCTGGTGCACCCCCAGTGGGTCCTCACAGCTGCCCACTGCATCAGGAACAAAAGCGTGATCTTGCTGGGTCGGCACAGCTTGTTTCATCCTGAAGACACAGGCCAGGTATTTCAGGTCAGCCACAGCTTCCCACACCCGCTCTACGATATGAGCCTCCTGAAGAATCGATTCCTCAGGCCAGGTGATGACTCCAGCCACGACCTCATGCTGCTCCGCCTGTCAGAGCCTGCCGAGCTCACGGATGCTGTGAAGGTCATGGACCTGCCCACCCAGGAGCCAGCACTGGGGACCACCTGCTACGCCTCAGGCTGGGGCAGCATTGAACCAGAGGAGTTCTTGACCCCAAAGAAACTTCAGTGTGTGGACCTCCATGTTATTTCCAATGACGTGTGTGCGCAAGTTCACCCTCAGAAGGTGACCAAGTTCATGCTGTGTGCTGGACGCTGGACAGGGGGCAAAAGCACCTGCTCGGGTGATTCTGGGGGCCCACTTGTCTGTAATGGTGTGCTTCAAGGTATCACGTCATGGGGCAGTGAACCATGTGCCCTGCCCGAAAGGCCTTCCCTGTACACCAAGGTGGTGCATTACCGGAAGTGGATCAAGGACACCATCGTGGCCAACCCCGGATCCCAGACCCTGAACTTTGATCTGCTGAAACTGGCAGGCGATGTGGAAAGCAACCCAGGCCCAATGGCAAGCGCGCGCCGCCCGCGCTGGCTGTGCGCTGGGGCGCTGGTGCTGGCGGGTGGCTTCTTTCTCCTCGGCTTCCTCTTCGGGTGGTTTATAAAATCCTCCAATGAAGCTACTAACATTACTCCAAAGCATAATATGAAAGCATTTTTGGATGAATTGAAAGCTGAGAACATCAAGAAGTTCTTATATAATTTTACACAGATACCACATTTAGCAGGAACAGAACAAAACTTTCAGCTTGCAAAGCAAATTCAATCCCAGTGGAAAGAATTTGGCCTGGATTCTGTTGAGCTGGCACATTATGATGTCCTGTTGTCCTACCCAAATAAGACTCATCCCAACTACATCTCAATAATTAATGAAGATGGAAATGAGATTTTCAACACATCATTATTTGAACCACCTCCTCCAGGATATGAAAATGTTTCGGATATTGTACCACCTTTCAGTGCTTTCTCTCCTCAAGGAATGCCAGAGGGCGATCTAGTGTATGTTAACTATGCACGAACTGAAGACTTCTTTAAATTGGAACGGGACATGAAAATCAATTGCTCTGGGAAAATTGTAATTGCCAGATATGGGAAAGTTTTCAGAGGAAATAAGGTTAAAAATGCCCAGCTGGCAGGGGCCAAAGGAGTCATTCTCTACTCCGACCCTGCTGACTACTTTGCTCCTGGGGTGAAGTCCTATCCAGATGGTTGGAATCTTCCTGGAGGTGGTGTCCAGCGTGGAAATATCCTAAATCTGAATGGTGCAGGAGACCCTCTCACACCAGGTTACCCAGCAAATGAATATGCTTATAGGCGTGGAATTGCAGAGGCTGTTGGTCTTCCAAGTATTCCTGTTCATCCAATTGGATACTATGATGCACAGAAGCTCCTAGAAAAAATGGGTGGCTCAGCACCACCAGATAGCAGCTGGAGAGGAAGTCTCAAAGTGCCCTACAATGTTGGACCTGGCTTTACTGGAAACTTTTCTACACAAAAAGTCAAGATGCACATCCACTCTACCAATGAAGTGACAAGAATTTACAATGTGATAGGTACTCTCAGAGGAGCAGTGGAACCAGACAGATATGTCATTCTGGGAGGTCACCGGGACTCATGGGTGTTTGGTGGTATTGACCCTCAGAGTGGAGCAGCTGTTGTTCATGAAATTGTGAGGAGCTTTGGAACACTGAAAAAGGAAGGGTGGAGACCTAGAAGAACAATTTTGTTTGCAAGCTGGGATGCAGAAGAATTTGGTCTTCTTGGTTCTACTGAGTGGGCAGAGGAGAATTCAAGACTCCTTCAAGAGCGTGGCGTGGCTTATATTAATGCTGACTCATCTATAGAAGGAAACTACACTCTGAGAGTTGATTGTACACCGCTGATGTACAGCTTGGTACACAACCTAACAAAAGAGCTGAAAAGCCCTGATGAAGGCTTTGAAGGCAAATCTCTTTATGAAAGTTGGACTAAAAAAAGTCCTTCCCCAGAGTTCAGTGGCATGCCCAGGATAAGCAAATTGGGATCTGGAAATGATTTTGAGGTGTTCTTCCAACGACTTGGAATTGCTTCAGGCAGAGCACGGTATACTAAAAATTGGGAAACAAACAAATTCAGCGGCTATCCACTGTATCACAGTGTCTATGAAACATATGAGTTGGTGGAAAAGTTTTATGATCCAATGTTTAAATATCACCTCACTGTGGCCCAGGTTCGAGGAGGGATGGTGTTTGAGCTGGCCAATTCCATAGTGCTCCCTTTTGATTGTCGAGATTATGCTGTAGTTTTAAGAAAGTATGCTGACAAAATCTACAGTATTTCTATGAAACATCCACAGGAAATGAAGACATACAGTGTATCATTTGATTCACTTTTTTCTGCAGTAAAGAATTTTACAGAAATTGCTTCCAAGTTCAGTGAGAGACTCCAGGACTTTGACAAAAGCAACCCAATAGTATTAAGAATGATGAATGATCAACTCATGTTTCTGGAAAGAGCATTTATTGATCCATTAGGGTTACCAGACAGGCCTTTTTATAGGCATGTCATCTATGCTCCAAGCAGCCACAACAAGTATGCAGGGGAGTCATTCCCAGGAATTTATGATGCTCTGTTTGATATTGAAAGCAAAGTGGACCCTTCCAAGGCCTGGGGAGAAGTGAAGAGACAGATTTATGTTGCAGCCTTCACAGTGCAGGCAGCTGCAGAGACTTTGAGTGAAGTAGCCTAAAGATCTGACCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCAGCAAGGCTGTGCTGCTTGCCCTGTTGATGGCAGGCTTGGCCCTGCAGCCAGGCACTGCCCTGCTGTGCTACTCCTGCAAAGCCCAGGTGAGCAACGAGGACTGCCTGCAGGTGGAGAACTGCACCCAGCTGGGGGAGCAGTGCTGGACCGCGCGCATCCGCGCAGTTGGCCTCCTGACCGTCATCAGCAAAGGCTGCAGCTTGAACTGCGTGGATGACTCACAGGACTACTACGTGGGCAAGAAGAACATCACGTGCTGTGACACCGACTTGTGCAACGCCAGCGGGGCCCATGCCCTGCAGCCGGCTGCCGCCATCCTTGCGCTGCTCCCTGCACTCGGCCTGCTGCTCTGGGGACCCGGCCAGCTATAGGGATCTGGGCCCTAACAAAACAAAAAGATGGGGTTATTCCCTAAACTTCATGGGTTACGTAATTGGAAGTTGGGGGACATTGCCACAAGATCATATTGTACAAAAGATCAAACACTGTTTTAGAAAACTTCCTGTAAACAGGCCTATTGATTGGAAAGTATGTCAAAGGATTGTGGGTCTTTTGGGCTTTGCTGCTCCATTTACACAATGTGGATATCCTGCCTTAATGCCTTTGTATGCATGTATACAAGCTAAACAGGCTTTCACTTTCTCGCCAACTTACAAGGCCTTTCTAAGTAAACAGTACATGAACCTTTACCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGGCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCCGGTCTGGAGCAAAGCTCATAGGAACTGACAATTCTGTCGTCCTCTCGCGGAAATATACATCGTTTCGATCTACGTATGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGAATTCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTC SEQ ID NO: 34. NUCLEOTIDESEQUENCE OF PLASMID 457GGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCAAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGGTCGACAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACTCACCGTCCGGATCTCAGCAAGCAGGTATGTACTCTCCAGGGTGGGCCTGGCTTCCCCAGTCAAGACTCCAGGGATTTGAGGGACGCTGTGGGCTCTTCTCTTACATGTACCTTTTGCTTGCCTCAACCCTGACTATCTTCCAGGTCAGGATCCCAGAGTCAGGGGTCTGTATTTTCCTGCTGGTGGCTCCAGTTCAGGAACAGTAAACCCTGCTCCGAATATTGCCTCTCACATCTCGTCAATCTCCGCGAGGACTGGGGACCCTGTGACGAACATGGCTAGCATTGTGGGAGGCTGGGAGTGCGAGAAGCATTCCCAACCCTGGCAGGTGCTTGTGGCCTCTCGTGGCAGGGCAGTCTGCGGCGGTGTTCTGGTGCACCCCCAGTGGGTCCTCACAGCTGCCCACTGCATCAGGAACAAAAGCGTGATCTTGCTGGGTCGGCACAGCTTGTTTCATCCTGAAGACACAGGCCAGGTATTTCAGGTCAGCCACAGCTTCCCACACCCGCTCTACGATATGAGCCTCCTGAAGAATCGATTCCTCAGGCCAGGTGATGACTCCAGCCACGACCTCATGCTGCTCCGCCTGTCAGAGCCTGCCGAGCTCACGGATGCTGTGAAGGTCATGGACCTGCCCACCCAGGAGCCAGCACTGGGGACCACCTGCTACGCCTCAGGCTGGGGCAGCATTGAACCAGAGGAGTTCTTGACCCCAAAGAAACTTCAGTGTGTGGACCTCCATGTTATTTCCAATGACGTGTGTGCGCAAGTTCACCCTCAGAAGGTGACCAAGTTCATGCTGTGTGCTGGACGCTGGACAGGGGGCAAAAGCACCTGCTCGGGTGATTCTGGGGGCCCACTTGTCTGTAATGGTGTGCTTCAAGGTATCACGTCATGGGGCAGTGAACCATGTGCCCTGCCCGAAAGGCCTTCCCTGTACACCAAGGTGGTGCATTACCGGAAGTGGATCAAGGACACCATCGTGGCCAACCCCGGATCCCAGACCCTGAACTTTGATCTGCTGAAACTGGCAGGCGATGTGGAAAGCAACCCAGGCCCAATGGCAAGCGCGCGCCGCCCGCGCTGGCTGTGCGCTGGGGCGCTGGTGCTGGCGGGTGGCTTCTTTCTCCTCGGCTTCCTCTTCGGGTGGTTTATAAAATCCTCCAATGAAGCTACTAACATTACTCCAAAGCATAATATGAAAGCATTTTTGGATGAATTGAAAGCTGAGAACATCAAGAAGTTCTTATATAATTTTACACAGATACCACATTTAGCAGGAACAGAACAAAACTTTCAGCTTGCAAAGCAAATTCAATCCCAGTGGAAAGAATTTGGCCTGGATTCTGTTGAGCTGGCACATTATGATGTCCTGTTGTCCTACCCAAATAAGACTCATCCCAACTACATCTCAATAATTAATGAAGATGGAAATGAGATTTTCAACACATCATTATTTGAACCACCTCCTCCAGGATATGAAAATGTTTCGGATATTGTACCACCTTTCAGTGCTTTCTCTCCTCAAGGAATGCCAGAGGGCGATCTAGTGTATGTTAACTATGCACGAACTGAAGACTTCTTTAAATTGGAACGGGACATGAAAATCAATTGCTCTGGGAAAATTGTAATTGCCAGATATGGGAAAGTTTTCAGAGGAAATAAGGTTAAAAATGCCCAGCTGGCAGGGGCCAAAGGAGTCATTCTCTACTCCGACCCTGCTGACTACTTTGCTCCTGGGGTGAAGTCCTATCCAGATGGTTGGAATCTTCCTGGAGGTGGTGTCCAGCGTGGAAATATCCTAAATCTGAATGGTGCAGGAGACCCTCTCACACCAGGTTACCCAGCAAATGAATATGCTTATAGGCGTGGAATTGCAGAGGCTGTTGGTCTTCCAAGTATTCCTGTTCATCCAATTGGATACTATGATGCACAGAAGCTCCTAGAAAAAATGGGTGGCTCAGCACCACCAGATAGCAGCTGGAGAGGAAGTCTCAAAGTGCCCTACAATGTTGGACCTGGCTTTACTGGAAACTTTTCTACACAAAAAGTCAAGATGCACATCCACTCTACCAATGAAGTGACAAGAATTTACAATGTGATAGGTACTCTCAGAGGAGCAGTGGAACCAGACAGATATGTCATTCTGGGAGGTCACCGGGACTCATGGGTGTTTGGTGGTATTGACCCTCAGAGTGGAGCAGCTGTTGTTCATGAAATTGTGAGGAGCTTTGGAACACTGAAAAAGGAAGGGTGGAGACCTAGAAGAACAATTTTGTTTGCAAGCTGGGATGCAGAAGAATTTGGTCTTCTTGGTTCTACTGAGTGGGCAGAGGAGAATTCAAGACTCCTTCAAGAGCGTGGCGTGGCTTATATTAATGCTGACTCATCTATAGAAGGAAACTACACTCTGAGAGTTGATTGTACACCGCTGATGTACAGCTTGGTACACAACCTAACAAAAGAGCTGAAAAGCCCTGATGAAGGCTTTGAAGGCAAATCTCTTTATGAAAGTTGGACTAAAAAAAGTCCTTCCCCAGAGTTCAGTGGCATGCCCAGGATAAGCAAATTGGGATCTGGAAATGATTTTGAGGTGTTCTTCCAACGACTTGGAATTGCTTCAGGCAGAGCACGGTATACTAAAAATTGGGAAACAAACAAATTCAGCGGCTATCCACTGTATCACAGTGTCTATGAAACATATGAGTTGGTGGAAAAGTTTTATGATCCAATGTTTAAATATCACCTCACTGTGGCCCAGGTTCGAGGAGGGATGGTGTTTGAGCTGGCCAATTCCATAGTGCTCCCTTTTGATTGTCGAGATTATGCTGTAGTTTTAAGAAAGTATGCTGACAAAATCTACAGTATTTCTATGAAACATCCACAGGAAATGAAGACATACAGTGTATCATTTGATTCACTTTTTTCTGCAGTAAAGAATTTTACAGAAATTGCTTCCAAGTTCAGTGAGAGACTCCAGGACTTTGACAAAAGCAACCCAATAGTATTAAGAATGATGAATGATCAACTCATGTTTCTGGAAAGAGCATTTATTGATCCATTAGGGTTACCAGACAGGCCTTTTTATAGGCATGTCATCTATGCTCCAAGCAGCCACAACAAGTATGCAGGGGAGTCATTCCCAGGAATTTATGATGCTCTGTTTGATATTGAAAGCAAAGTGGACCCTTCCAAGGCCTGGGGAGAAGTGAAGAGACAGATTTATGTTGCAGCCTTCACAGTGCAGGCAGCTGCAGAGACTTTGAGTGAAGTAGCCGGATCCGAAGGTAGGGGTTCATTATTGACCTGTGGAGATGTCGAAGAAAACCCAGGACCCGCAAGCAAGGCTGTGCTGCTTGCCCTGTTGATGGCAGGCTTGGCCCTGCAGCCAGGCACTGCCCTGCTGTGCTACTCCTGCAAAGCCCAGGTGAGCAACGAGGACTGCCTGCAGGTGGAGAACTGCACCCAGCTGGGGGAGCAGTGCTGGACCGCGCGCATCCGCGCAGTTGGCCTCCTGACCGTCATCAGCAAAGGCTGCAGCTTGAACTGCGTGGATGACTCACAGGACTACTACGTGGGCAAGAAGAACATCACGTGCTGTGACACCGACTTGTGCAACGCCAGCGGGGCCCATGCCCTGCAGCCGGCTGCCGCCATCCTTGCGCTGCTCCCTGCACTCGGCCTGCTGCTCTGGGGACCCGGCCAGCTATAGAGATCTGGGCCCTAACAAAACAAAAAGATGGGGTTATTCCCTAAACTTCATGGGTTACGTAATTGGAAGTTGGGGGACATTGCCACAAGATCATATTGTACAAAAGATCAAACACTGTTTTAGAAAACTTCCTGTAAACAGGCCTATTGATTGGAAAGTATGTCAAAGGATTGTGGGTCTTTTGGGCTTTGCTGCTCCATTTACACAATGTGGATATCCTGCCTTAATGCCTTTGTATGCATGTATACAAGCTAAACAGGCTTTCACTTTCTCGCCAACTTACAAGGCCTTTCTAAGTAAACAGTACATGAACCTTTACCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGGCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCCGGTCTGGAGCAAAGCTCATAGGAACTGACAATTCTGTCGTCCTCTCGCGGAAATATACATCGTTTCGATCTACGTATGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGAATTCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTC SEQ ID NO: 35. NUCLEOTIDE SEQUENCE OF PLASMID 458GGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCAAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGGTCGACAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACTCACCGTCCGGATCTCAGCAAGCAGGTATGTACTCTCCAGGGTGGGCCTGGCTTCCCCAGTCAAGACTCCAGGGATTTGAGGGACGCTGTGGGCTCTTCTCTTACATGTACCTTTTGCTTGCCTCAACCCTGACTATCTTCCAGGTCAGGATCCCAGAGTCAGGGGTCTGTATTTTCCTGCTGGTGGCTCCAGTTCAGGAACAGTAAACCCTGCTCCGAATATTGCCTCTCACATCTCGTCAATCTCCGCGAGGACTGGGGACCCTGTGACGAACATGGCTAGCATTGTGGGAGGCTGGGAGTGCGAGAAGCATTCCCAACCCTGGCAGGTGCTTGTGGCCTCTCGTGGCAGGGCAGTCTGCGGCGGTGTTCTGGTGCACCCCCAGTGGGTCCTCACAGCTGCCCACTGCATCAGGAACAAAAGCGTGATCTTGCTGGGTCGGCACAGCTTGTTTCATCCTGAAGACACAGGCCAGGTATTTCAGGTCAGCCACAGCTTCCCACACCCGCTCTACGATATGAGCCTCCTGAAGAATCGATTCCTCAGGCCAGGTGATGACTCCAGCCACGACCTCATGCTGCTCCGCCTGTCAGAGCCTGCCGAGCTCACGGATGCTGTGAAGGTCATGGACCTGCCCACCCAGGAGCCAGCACTGGGGACCACCTGCTACGCCTCAGGCTGGGGCAGCATTGAACCAGAGGAGTTCTTGACCCCAAAGAAACTTCAGTGTGTGGACCTCCATGTTATTTCCAATGACGTGTGTGCGCAAGTTCACCCTCAGAAGGTGACCAAGTTCATGCTGTGTGCTGGACGCTGGACAGGGGGCAAAAGCACCTGCTCGGGTGATTCTGGGGGCCCACTTGTCTGTAATGGTGTGCTTCAAGGTATCACGTCATGGGGCAGTGAACCATGTGCCCTGCCCGAAAGGCCTTCCCTGTACACCAAGGTGGTGCATTACCGGAAGTGGATCAAGGACACCATCGTGGCCAACCCCGGATCCGAAGGTAGGGGTTCATTATTGACCTGTGGAGATGTCGAAGAAAACCCAGGACCCGCTAGCAAGGCTGTGCTGCTTGCCCTGTTGATGGCAGGCTTGGCCCTGCAGCCAGGCACTGCCCTGCTGTGCTACTCCTGCAAAGCCCAGGTGAGCAACGAGGACTGCCTGCAGGTGGAGAACTGCACCCAGCTGGGGGAGCAGTGCTGGACCGCGCGCATCCGCGCAGTTGGCCTCCTGACCGTCATCAGCAAAGGCTGCAGCTTGAACTGCGTGGATGACTCACAGGACTACTACGTGGGCAAGAAGAACATCACGTGCTGTGACACCGACTTGTGCAACGCCAGCGGGGCCCATGCCCTGCAGCCGGCTGCCGCCATCCTTGCGCTGCTCCCTGCACTCGGCCTGCTGCTCTGGGGACCCGGCCAGCTAGGATCCCAGACCCTGAACTTTGATCTGCTGAAACTGGCAGGCGATGTGGAAAGCAACCCAGGCCCAATGGCAAGCGCGCGCCGCCCGCGCTGGCTGTGCGCTGGGGCGCTGGTGCTGGCGGGTGGCTTCTTTCTCCTCGGCTTCCTCTTCGGGTGGTTTATAAAATCCTCCAATGAAGCTACTAACATTACTCCAAAGCATAATATGAAAGCATTTTTGGATGAATTGAAAGCTGAGAACATCAAGAAGTTCTTATATAATTTTACACAGATACCACATTTAGCAGGAACAGAACAAAACTTTCAGCTTGCAAAGCAAATTCAATCCCAGTGGAAAGAATTTGGCCTGGATTCTGTTGAGCTGGCACATTATGATGTCCTGTTGTCCTACCCAAATAAGACTCATCCCAACTACATCTCAATAATTAATGAAGATGGAAATGAGATTTTCAACACATCATTATTTGAACCACCTCCTCCAGGATATGAAAATGTTTCGGATATTGTACCACCTTTCAGTGCTTTCTCTCCTCAAGGAATGCCAGAGGGCGATCTAGTGTATGTTAACTATGCACGAACTGAAGACTTCTTTAAATTGGAACGGGACATGAAAATCAATTGCTCTGGGAAAATTGTAATTGCCAGATATGGGAAAGTTTTCAGAGGAAATAAGGTTAAAAATGCCCAGCTGGCAGGGGCCAAAGGAGTCATTCTCTACTCCGACCCTGCTGACTACTTTGCTCCTGGGGTGAAGTCCTATCCAGATGGTTGGAATCTTCCTGGAGGTGGTGTCCAGCGTGGAAATATCCTAAATCTGAATGGTGCAGGAGACCCTCTCACACCAGGTTACCCAGCAAATGAATATGCTTATAGGCGTGGAATTGCAGAGGCTGTTGGTCTTCCAAGTATTCCTGTTCATCCAATTGGATACTATGATGCACAGAAGCTCCTAGAAAAAATGGGTGGCTCAGCACCACCAGATAGCAGCTGGAGAGGAAGTCTCAAAGTGCCCTACAATGTTGGACCTGGCTTTACTGGAAACTTTTCTACACAAAAAGTCAAGATGCACATCCACTCTACCAATGAAGTGACAAGAATTTACAATGTGATAGGTACTCTCAGAGGAGCAGTGGAACCAGACAGATATGTCATTCTGGGAGGTCACCGGGACTCATGGGTGTTTGGTGGTATTGACCCTCAGAGTGGAGCAGCTGTTGTTCATGAAATTGTGAGGAGCTTTGGAACACTGAAAAAGGAAGGGTGGAGACCTAGAAGAACAATTTTGTTTGCAAGCTGGGATGCAGAAGAATTTGGTCTTCTTGGTTCTACTGAGTGGGCAGAGGAGAATTCAAGACTCCTTCAAGAGCGTGGCGTGGCTTATATTAATGCTGACTCATCTATAGAAGGAAACTACACTCTGAGAGTTGATTGTACACCGCTGATGTACAGCTTGGTACACAACCTAACAAAAGAGCTGAAAAGCCCTGATGAAGGCTTTGAAGGCAAATCTCTTTATGAAAGTTGGACTAAAAAAAGTCCTTCCCCAGAGTTCAGTGGCATGCCCAGGATAAGCAAATTGGGATCTGGAAATGATTTTGAGGTGTTCTTCCAACGACTTGGAATTGCTTCAGGCAGAGCACGGTATACTAAAAATTGGGAAACAAACAAATTCAGCGGCTATCCACTGTATCACAGTGTCTATGAAACATATGAGTTGGTGGAAAAGTTTTATGATCCAATGTTTAAATATCACCTCACTGTGGCCCAGGTTCGAGGAGGGATGGTGTTTGAGCTGGCCAATTCCATAGTGCTCCCTTTTGATTGTCGAGATTATGCTGTAGTTTTAAGAAAGTATGCTGACAAAATCTACAGTATTTCTATGAAACATCCACAGGAAATGAAGACATACAGTGTATCATTTGATTCACTTTTTTCTGCAGTAAAGAATTTTACAGAAATTGCTTCCAAGTTCAGTGAGAGACTCCAGGACTTTGACAAAAGCAACCCAATAGTATTAAGAATGATGAATGATCAACTCATGTTTCTGGAAAGAGCATTTATTGATCCATTAGGGTTACCAGACAGGCCTTTTTATAGGCATGTCATCTATGCTCCAAGCAGCCACAACAAGTATGCAGGGGAGTCATTCCCAGGAATTTATGATGCTCTGTTTGATATTGAAAGCAAAGTGGACCCTTCCAAGGCCTGGGGAGAAGTGAAGAGACAGATTTATGTTGCAGCCTTCACAGTGCAGGCAGCTGCAGAGACTTTGAGTGAAGTAGCCTAAAGATCTGGGCCCTAACAAAACAAAAAGATGGGGTTATTCCCTAAACTTCATGGGTTACGTAATTGGAAGTTGGGGGACATTGCCACAAGATCATATTGTACAAAAGATCAAACACTGTTTTAGAAAACTTCCTGTAAACAGGCCTATTGATTGGAAAGTATGTCAAAGGATTGTGGGTCTTTTGGGCTTTGCTGCTCCATTTACACAATGTGGATATCCTGCCTTAATGCCTTTGTATGCATGTATACAAGCTAAACAGGCTTTCACTTTCTCGCCAACTTACAAGGCCTTTCTAAGTAAACAGTACATGAACCTTTACCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGGCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCCGGTCTGGAGCAAAGCTCATAGGAACTGACAATTCTGTCGTCCTCTCGCGGAAATATACATCGTTTCGATCTACGTATGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGAATTCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTC SEQ ID NO: 36. NUCLEOTIDE SEQUENCE OF PLASMID 459GGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCAAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGGTCGACAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACTCACCGTCCGGATCTCAGCAAGCAGGTATGTACTCTCCAGGGTGGGCCTGGCTTCCCCAGTCAAGACTCCAGGGATTTGAGGGACGCTGTGGGCTCTTCTCTTACATGTACCTTTTGCTTGCCTCAACCCTGACTATCTTCCAGGTCAGGATCCCAGAGTCAGGGGTCTGTATTTTCCTGCTGGTGGCTCCAGTTCAGGAACAGTAAACCCTGCTCCGAATATTGCCTCTCACATCTCGTCAATCTCCGCGAGGACTGGGGACCCTGTGACGAACATGGCTAGCAAGGCTGTGCTGCTTGCCCTGTTGATGGCAGGCTTGGCCCTGCAGCCAGGCACTGCCCTGCTGTGCTACTCCTGCAAAGCCCAGGTGAGCAACGAGGACTGCCTGCAGGTGGAGAACTGCACCCAGCTGGGGGAGCAGTGCTGGACCGCGCGCATCCGCGCAGTTGGCCTCCTGACCGTCATCAGCAAAGGCTGCAGCTTGAACTGCGTGGATGACTCACAGGACTACTACGTGGGCAAGAAGAACATCACGTGCTGTGACACCGACTTGTGCAACGCCAGCGGGGCCCATGCCCTGCAGCCGGCTGCCGCCATCCTTGCGCTGCTCCCTGCACTCGGCCTGCTGCTCTGGGGACCCGGCCAGCTAGGATCCCAGACCCTGAACTTTGATCTGCTGAAACTGGCAGGCGATGTGGAAAGCAACCCAGGCCCAATGGCAAGCGCGCGCCGCCCGCGCTGGCTGTGCGCTGGGGCGCTGGTGCTGGCGGGTGGCTTCTTTCTCCTCGGCTTCCTCTTCGGGTGGTTTATAAAATCCTCCAATGAAGCTACTAACATTACTCCAAAGCATAATATGAAAGCATTTTTGGATGAATTGAAAGCTGAGAACATCAAGAAGTTCTTATATAATTTTACACAGATACCACATTTAGCAGGAACAGAACAAAACTTTCAGCTTGCAAAGCAAATTCAATCCCAGTGGAAAGAATTTGGCCTGGATTCTGTTGAGCTGGCACATTATGATGTCCTGTTGTCCTACCCAAATAAGACTCATCCCAACTACATCTCAATAATTAATGAAGATGGAAATGAGATTTTCAACACATCATTATTTGAACCACCTCCTCCAGGATATGAAAATGTTTCGGATATTGTACCACCTTTCAGTGCTTTCTCTCCTCAAGGAATGCCAGAGGGCGATCTAGTGTATGTTAACTATGCACGAACTGAAGACTTCTTTAAATTGGAACGGGACATGAAAATCAATTGCTCTGGGAAAATTGTAATTGCCAGATATGGGAAAGTTTTCAGAGGAAATAAGGTTAAAAATGCCCAGCTGGCAGGGGCCAAAGGAGTCATTCTCTACTCCGACCCTGCTGACTACTTTGCTCCTGGGGTGAAGTCCTATCCAGATGGTTGGAATCTTCCTGGAGGTGGTGTCCAGCGTGGAAATATCCTAAATCTGAATGGTGCAGGAGACCCTCTCACACCAGGTTACCCAGCAAATGAATATGCTTATAGGCGTGGAATTGCAGAGGCTGTTGGTCTTCCAAGTATTCCTGTTCATCCAATTGGATACTATGATGCACAGAAGCTCCTAGAAAAAATGGGTGGCTCAGCACCACCAGATAGCAGCTGGAGAGGAAGTCTCAAAGTGCCCTACAATGTTGGACCTGGCTTTACTGGAAACTTTTCTACACAAAAAGTCAAGATGCACATCCACTCTACCAATGAAGTGACAAGAATTTACAATGTGATAGGTACTCTCAGAGGAGCAGTGGAACCAGACAGATATGTCATTCTGGGAGGTCACCGGGACTCATGGGTGTTTGGTGGTATTGACCCTCAGAGTGGAGCAGCTGTTGTTCATGAAATTGTGAGGAGCTTTGGAACACTGAAAAAGGAAGGGTGGAGACCTAGAAGAACAATTTTGTTTGCAAGCTGGGATGCAGAAGAATTTGGTCTTCTTGGTTCTACTGAGTGGGCAGAGGAGAATTCAAGACTCCTTCAAGAGCGTGGCGTGGCTTATATTAATGCTGACTCATCTATAGAAGGAAACTACACTCTGAGAGTTGATTGTACACCGCTGATGTACAGCTTGGTACACAACCTAACAAAAGAGCTGAAAAGCCCTGATGAAGGCTTTGAAGGCAAATCTCTTTATGAAAGTTGGACTAAAAAAAGTCCTTCCCCAGAGTTCAGTGGCATGCCCAGGATAAGCAAATTGGGATCTGGAAATGATTTTGAGGTGTTCTTCCAACGACTTGGAATTGCTTCAGGCAGAGCACGGTATACTAAAAATTGGGAAACAAACAAATTCAGCGGCTATCCACTGTATCACAGTGTCTATGAAACATATGAGTTGGTGGAAAAGTTTTATGATCCAATGTTTAAATATCACCTCACTGTGGCCCAGGTTCGAGGAGGGATGGTGTTTGAGCTGGCCAATTCCATAGTGCTCCCTTTTGATTGTCGAGATTATGCTGTAGTTTTAAGAAAGTATGCTGACAAAATCTACAGTATTTCTATGAAACATCCACAGGAAATGAAGACATACAGTGTATCATTTGATTCACTTTTTTCTGCAGTAAAGAATTTTACAGAAATTGCTTCCAAGTTCAGTGAGAGACTCCAGGACTTTGACAAAAGCAACCCAATAGTATTAAGAATGATGAATGATCAACTCATGTTTCTGGAAAGAGCATTTATTGATCCATTAGGGTTACCAGACAGGCCTTTTTATAGGCATGTCATCTATGCTCCAAGCAGCCACAACAAGTATGCAGGGGAGTCATTCCCAGGAATTTATGATGCTCTGTTTGATATTGAAAGCAAAGTGGACCCTTCCAAGGCCTGGGGAGAAGTGAAGAGACAGATTTATGTTGCAGCCTTCACAGTGCAGGCAGCTGCAGAGACTTTGAGTGAAGTAGCCTAAAGATCTGACCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCAGCATTGTGGGAGGCTGGGAGTGCGAGAAGCATTCCCAACCCTGGCAGGTGCTTGTGGCCTCTCGTGGCAGGGCAGTCTGCGGCGGTGTTCTGGTGCACCCCCAGTGGGTCCTCACAGCTGCCCACTGCATCAGGAACAAAAGCGTGATCTTGCTGGGTCGGCACAGCTTGTTTCATCCTGAAGACACAGGCCAGGTATTTCAGGTCAGCCACAGCTTCCCACACCCGCTCTACGATATGAGCCTCCTGAAGAATCGATTCCTCAGGCCAGGTGATGACTCCAGCCACGACCTCATGCTGCTCCGCCTGTCAGAGCCTGCCGAGCTCACGGATGCTGTGAAGGTCATGGACCTGCCCACCCAGGAGCCAGCACTGGGGACCACCTGCTACGCCTCAGGCTGGGGCAGCATTGAACCAGAGGAGTTCTTGACCCCAAAGAAACTTCAGTGTGTGGACCTCCATGTTATTTCCAATGACGTGTGTGCGCAAGTTCACCCTCAGAAGGTGACCAAGTTCATGCTGTGTGCTGGACGCTGGACAGGGGGCAAAAGCACCTGCTCGGGTGATTCTGGGGGCCCACTTGTCTGTAATGGTGTGCTTCAAGGTATCACGTCATGGGGCAGTGAACCATGTGCCCTGCCCGAAAGGCCTTCCCTGTACACCAAGGTGGTGCATTACCGGAAGTGGATCAAGGACACCATCGTGGCCAACCCCTGAGGATCTGGGCCCTAACAAAACAAAAAGATGGGGTTATTCCCTAAACTTCATGGGTTACGTAATTGGAAGTTGGGGGACATTGCCACAAGATCATATTGTACAAAAGATCAAACACTGTTTTAGAAAACTTCCTGTAAACAGGCCTATTGATTGGAAAGTATGTCAAAGGATTGTGGGTCTTTTGGGCTTTGCTGCTCCATTTACACAATGTGGATATCCTGCCTTAATGCCTTTGTATGCATGTATACAAGCTAAACAGGCTTTCACTTTCTCGCCAACTTACAAGGCCTTTCTAAGTAAACAGTACATGAACCTTTACCCCGTTGCTCGGCAACGGCCTGGTCTGTGCCAAGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCTTGGCCATAGGCCATCAGCGCATGCGTGGAACCTTTGTGGCTCCTCTGCCGATCCATACTGCGGAACTCCTAGCCGCTTGTTTTGCTCGCAGCCGGTCTGGAGCAAAGCTCATAGGAACTGACAATTCTGTCGTCCTCTCGCGGAAATATACATCGTTTCGATCTACGTATGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGAATTCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTC SEQ ID NO: 37. NUCLEOTIDE SEQUENCEOF PSHUTTLE IRESCATCATCAATAATATACCTTATTTTGGATTGAAGCCAATATGATAATGAGGGGGTGGAGTTTGTGACGTGGCGCGGGGCGTGGGAACGGGGCGGGTGACGTAGTAGTGTGGCGGAAGTGTGATGTTGCAAGTGTGGCGGAACACATGTAAGCGACGGATGTGGCAAAAGTGACGTTTTTGGTGTGCGCCGGTGTACACAGGAAGTGACAATTTTCGCGCGGTTTTAGGCGGATGTTGTAGTAAATTTGGGCGTAACCGAGTAAGATTTGGCCATTTTCGCGGGAAAACTGAATAAGAGGAAGTGAAATCTGAATAATTTTGTGTTACTCATAGCGCGTAATACTGTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCCGCTAGAGATCCACCATGGCTAGCGGTGCCCCGACGTTGCCCCCTGCCTGGCAGCCCTTTCTCAAGGACCACCGCATCTCTACATTCAAGAACTGGCCCTTCTTGGAGGGCTGCGCCTGCGCCCCGGAGCGGATGGCCGAGGCTGGCTTCATCCACTGCCCCACTGAGAACGAGCCAGACTTGGCCCAGTGTTTCTTCTGCTTCAAGGAGCTGGAAGGCTGGGAGCCAGATGACGACCCCATAGAGGAACATAAAAAGCATTCGTCCGGTTGCGCTTTCCTTTCTGTCAAGAAGCAGTTTGAAGAATTAACCCTTGGTGAATTTTTGAAACTGGACAGAGAAAGAGCCAAGAACAAAATTGCAAAGGAAACCAACAATAAGAAGAAAGAATTTGAGGAAACTGCGGAGAAAGTGCGCCGTGCCATCGAGCAGCTGGCTGCCATGGATTAGAGATCTGACCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATAATATGGCGGCCGCTCGAGCCTAAGCTTCTAGATAAGATATCCGATCCACCGGATCTAGATAACTGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTAACGCGGATCTGGGCGTGGTTAAGGGTGGGAAAGAATATATAAGGTGGGGGTCTTATGTAGTTTTGTATCTGTTTTGCAGCAGCCGCCGCCGCCATGAGCACCAACTCGTTTGATGGAAGCATTGTGAGCTCATATTTGACAACGCGCATGCCCCCATGGGCCGGGGTGCGTCAGAATGTGATGGGCTCCAGCATTGATGGTCGCCCCGTCCTGCCCGCAAACTCTACTACCTTGACCTACGAGACCGTGTCTGGAACGCCGTTGGAGACTGCAGCCTCCGCCGCCGCTTCAGCCGCTGCAGCCACCGCCCGCGGGATTGTGACTGACTTTGCTTTCCTGAGCCCGCTTGCAAGCAGTGCAGCTTCCCGTTCATCCGCCCGCGATGACAAGTTGACGGCTCTTTTGGCACAATTGGATTCTTTGACCCGGGAACTTAATGTCGTTTCTCAGCAGCTGTTGGATCTGCGCCAGCAGGTTTCTGCCCTGAAGGCTTCCTCCCCTCCCAATGCGGTTTAAAACATAAATAAAAAACCAGACTCTGTTTGGATTTGGATCAAGCAAGTGTCTTGCTGTCTTTATTTAGGGGTTTTGCGCGCGCGGTAGGCCCGGGACCAGCGGTCTCGGTCGTTGAGGGTCCTGTGTATTTTTTCCAGGACGTGGTAAAGGTGACTCTGGATGTTCAGATACATGGGCATAAGCCCGTCTCTGGGGTGGAGGTAGCACCACTGCAGAGCTTCATGCTGCGGGGTGGTGTTGTAGATGATCCAGTCGTAGCAGGAGCGCTGGGCGTGGTGCCTAAAAATGTCTTTCAGTAGCAAGCTGATTGCCAGGGGCAGGCCCTTGGTGTAAGTGTTTACAAAGCGGTTAAGCTGGGATGGGTGCATACGTGGGGATATGAGATGCATCTTGGACTGTATTTTTAGGTTGGCTATGTTCCCAGCCATATCCCTCCGGGGATTCATGTTGTGCAGAACCACCAGCACAGTGTATCCGGTGCACTTGGGAAATTTGTCATGTAGCTTAGAAGGAAATGCGTGGAAGAACTTGGAGACGCCCTTGTGACCTCCAAGATTTTCCATGCATTCGTCCATAATGATGGCAATGGGCCCACGGGCGGCGGCCTGGGCGAAGATATTTCTGGGATCACTAACGTCATAGTTGTGTTCCAGGATGAGATCGTCATAGGCCATTTTTACAAAGCGCGGGCGGAGGGTGCCAGACTGCGGTATAATGGTTCCATCCGGCCCAGGGGCGTAGTTACCCTCACAGATTTGCATTTCCCACGCTTTGAGTTCAGATGGGGGGATCATGTCTACCTGCGGGGCGATGAAGAAAACGGTTTCCGGGGTAGGGGAGATCAGCTGGGAAGAAAGCAGGTTCCTGAGCAGCTGCGACTTACCGCAGCCGGTGGGCCCGTAAATCACACCTATTACCGGCTGCAACTGGTAGTTAAGAGAGCTGCAGCTGCCGTCATCCCTGAGCAGGGGGGCCACTTCGTTAAGCATGTCCCTGACTCGCATGTTTTCCCTGACCAAATCCGCCAGAAGGCGCTCGCCGCCCAGCGATAGCAGTTCTTGCAAGGAAGCAAAGTTTTTCAACGGTTTGAGACCGTCCGCCGTAGGCATGCTTTTGAGCGTTTGACCAAGCAGTTCCAGGCGGTCCCACAGCTCGGTCACCTGCTCTACGGCATCTCGATCCAGCATATCTCCTCGTTTCGCGGGTTGGGGCGGCTTTCGCTGTACGGCAGTAGTCGGTGCTCGTCCAGACGGGCCAGGGTCATGTCTTTCCACGGGCGCAGGGTCCTCGTCAGCGTAGTCTGGGTCACGGTGAAGGGGTGCGCTCCGGGCTGCGCGCTGGCCAGGGTGCGCTTGAGGCTGGTCCTGCTGGTGCTGAAGCGCTGCCGGTCTTCGCCCTGCGCGTCGGCCAGGTAGCATTTGACCATGGTGTCATAGTCCAGCCCCTCCGCGGCGTGGCCCTTGGCGCGCAGCTTGCCCTTGGAGGAGGCGCCGCACGAGGGGCAGTGCAGACTTTTGAGGGCGTAGAGCTTGGGCGCGAGAAATACCGATTCCGGGGAGTAGGCATCCGCGCCGCAGGCCCCGCAGACGGTCTCGCATTCCACGAGCCAGGTGAGCTCTGGCCGTTCGGGGTCAAAAACCAGGTTTCCCCCATGCTTTTTGATGCGTTTCTTACCTCTGGTTTCCATGAGCCGGTGTCCACGCTCGGTGACGAAAAGGCTGTCCGTGTCCCCGTATACAGACTTGAGAGGGAGTTTAAACGAATTCAATAGCTTGTTGCATGGGCGGCGATATAAAATGCAAGGTGCTGCTCAAAAAATCAGGCAAAGCCTCGCGCAAAAAAGAAAGCACATCGTAGTCATGCTCATGCAGATAAAGGCAGGTAAGCTCCGGAACCACCACAGAAAAAGACACCATTTTTCTCTCAAACATGTCTGCGGGTTTCTGCATAAACACAAAATAAAATAACAAAAAAACATTTAAACATTAGAAGCCTGTCTTACAACAGGAAAAACAACCCTTATAAGCATAAGACGGACTACGGCCATGCCGGCGTGACCGTAAAAAAACTGGTCACCGTGATTAAAAAGCACCACCGACAGCTCCTCGGTCATGTCCGGAGTCATAATGTAAGACTCGGTAAACACATCAGGTTGATTCACATCGGTCAGTGCTAAAAAGCGACCGAAATAGCCCGGGGGAATACATACCCGCAGGCGTAGAGACAACATTACAGCCCCCATAGGAGGTATAACAAAATTAATAGGAGAGAAAAACACATAAACACCTGAAAAACCCTCCTGCCTAGGCAAAATAGCACCCTCCCGCTCCAGAACAACATACAGCGCTTCCACAGCGGCAGCCATAACAGTCAGCCTTACCAGTAAAAAAGAAAACCTATTAAAAAAACACCACTCGACACGGCACCAGCTCAATCAGTCACAGTGTAAAAAAGGGCCAAGTGCAGAGCGAGTATATATAGGACTAAAAAATGACGTAACGGTTAAAGTCCACAAAAAACACCCAGAAAACCGCACGCGAACCTACGCCCAGAAACGAAAGCCAAAAAACCCACAACTTCCTCAAATCGTCACTTCCGTTTTCCCACGTTACGTCACTTCCCATTTTAAGAAAACTACAATTCCCAACACATACAAGTTACTCCGCCCTAAAACCTACGTCACCCGCCCCGTTCCCACGCCCCGCGCCACGTCACAAACTCCACCCCCTCATTATCATATTGGCTTCAATCCAAAATAAGGTATATTATTGATGATGTTAATTAACATGCATGGATCCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGCCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTCACGTAGAAAGCCAGTCCGCAGAAACGGTGCTGACCCCGGATGAATGTCAGCTACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCAGTGGGCTTACATGGCGATAGCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGCTCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAATTTTGTTAAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCACCATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGTGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCGCGCTTAATGCGCCGCTACAGGGCGCGTCCATTCGCCATTCAGGATCGAATTAATTCTTAATTAA SEQ ID NO: 38. Amino acidsequence of Her-2 antigen:MASELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLDSVAPAAGATPGGLQELQLRSLTEILKGGVLIRRSPQLCHQDTVLWEDVFRKNNQLALVLMDTNRSRACHPCAPMCKANHCWGESSQDCQTLTRTICTSACARCKAPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREARAITSANVQDFVGCKKIFGSLAFLPESFDGDPASGTAPLQPEQLQVFETLEEITGYLYISAWPDSFPNLSVFQNLRVIRGRILHNGAYSLTLQGLGISWLGLRSLQELGSGLALVHRNARLCFVHTVPWDQLFRNPHQALLHSGNRPEEDCVGEGFVCYSLCAHGHCWGPGPTQCVNCSHFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRNEDLGPSSPMDSTFYRSLLEDEDMGELVDAEEYLVPQQGFFCPDPTPGTGSTAHRRHRSSSARNGGGDLTLGMEPSGEGPPRSPRAPSEGTGSDVFDGDLAVGVTKGLQSLSPQDLSPLQRYSEDPTLPLPSETDGKVAPLSCSPQPEFVNQSDVQPKSPLTPEGPPSPARPTGATLERAKTLSPGKNGVVKDVFTFGGAVENPEFLAPREGTASPPHPSPAFSPAFDNLFFWDQNSSEQGPPPSNFEGTPTAENPEFLGLDVPV (signal sequenceunderlined) SEQ ID NO: 39. Nucleic acid sequence encoding the Her-2antigen amino acid sequence of SEQ ID NO: 38ATGGCTAGCGAGCTGGCCGCCCTGTGTAGATGGGGACTGCTGCTGGCTCTGCTGCCTCCTGGAGCCGCTTCTACACAGGTCTGCACCGGCACCGACATGAAGCTGAGACTGCCCGCCAGCCCCGAGACACACCTGGACATGCTGCGGCACCTGTACCAGGGCTGCCAGGTGGTCCAGGGGAATCTGGAACTGACCTACCTGCCCACCAACGCCAGCCTGAGCTTCCTGCAGGACATCCAGGAAGTGCAGGGCTACGTCCTGATCGCCCACAACCAGGTCCGCCAGGTGCCCCTGCAGCGGCTGAGAATCGTGCGGGGCACCCAGCTGTTCGAGGACAACTACGCCCTGGCCGTGCTGGACAACGGCGACCCTCTGGATAGCGTGGCCCCTGCTGCTGGGGCTACACCTGGCGGACTGCAGGAACTGCAGCTGCGGAGCCTGACCGAGATCCTGAAGGGCGGCGTGCTGATCAGGCGGAGCCCTCAGCTGTGCCACCAGGACACCGTGCTGTGGGAGGACGTGTTCCGGAAGAACAACCAGCTGGCCCTCGTGCTGATGGACACCAACAGAAGCCGGGCCTGCCACCCCTGCGCCCCCATGTGCAAGGCCAATCACTGCTGGGGAGAGAGCAGCCAGGACTGCCAGACCCTGACCCGGACCATCTGCACCAGCGCCTGCGCCAGATGCAAGGCCCCCCTGCCTACCGACTGCTGCCACGAACAGTGCGCCGCTGGCTGCACCGGCCCCAAGCACAGCGATTGCCTGGCCTGCCTGCACTTCAACCACAGCGGCATCTGCGAGCTGCACTGCCCTGCCCTGGTGACATACAACACCGACACCTTCGAGAGCATGCCCAACCCCGAGGGCCGGTACACCTTCGGCGCCAGCTGTGTGACCGCCTGCCCCTACAACTACCTGAGCACCGACGTGGGCAGCTGCACCCTGGTGTGCCCCCTGCACAACCAGGAAGTGACCGCCGAGGACGGCACCCAGAGATGCGAGAAGTGCAGCAAGCCTTGCGCCAGAGTGTGCTACGGCCTGGGCATGGAACACCTGAGAGAGGCCAGAGCCATCACCAGCGCCAACGTGCAGGACTTCGTGGGCTGCAAGAAGATTTTCGGCTCCCTGGCCTTCCTGCCCGAGAGCTTCGACGGCGATCCTGCCTCTGGCACCGCCCCTCTGCAGCCTGAGCAGCTGCAGGTCTTCGAGACACTGGAAGAGATCACCGGCTACCTGTACATCAGCGCCTGGCCCGACAGCTTCCCCAACCTGAGCGTGTTCCAGAACCTGAGAGTGATCCGGGGCAGAATCCTGCACAACGGCGCCTACAGCCTGACCCTGCAGGGCCTGGGAATCAGCTGGCTGGGCCTGCGGAGCCTGCAGGAACTGGGATCTGGCCTGGCTCTGGTGCACCGGAACGCCCGGCTGTGCTTCGTGCACACCGTGCCCTGGGACCAGCTGTTCAGAAACCCCCACCAGGCTCTGCTGCACAGCGGCAACCGGCCCGAAGAGGATTGCGTGGGCGAGGGCTTCGTGTGCTACTCCCTGTGCGCCCACGGCCACTGTTGGGGACCTGGCCCTACCCAGTGCGTGAACTGCAGCCACTTCCTGCGGGGCCAAGAATGCGTGGAAGAGTGCCGGGTGCTGCAGGGACTGCCCCGGGAATACGTGAACGCCAGACACTGCCTGCCTTGCCACCCCGAGTGCCAGCCCCAGAATGGCAGCGTGACCTGCTTCGGACCCGAGGCCGATCAGTGTGTGGCCTGCGCCCACTACAAGGACCCCCCATTCTGCGTGGCCAGATGCCCCAGCGGCGTGAAGCCCGACCTGAGCTACATGCCCATCTGGAAGTTCCCCGACGAGGAAGGCGCCTGCCAGCCTTGCCCCATCAACTGCACCCACAGCTGCGTGGACCTGGACGACAAGGGCTGCCCTGCCGAGCAGAGAGCCAGCCCCCTGACCAGCATCATCAGCGCCGTGGTGGGAATCCTGCTGGTGGTGGTGCTGGGCGTGGTGTTCGGCATCCTGATCAAGCGGCGGCAGCAGAAGATCCGGAAGTACACCATGCGGCGGAACGAGGACCTGGGCCCCTCTAGCCCCATGGACAGCACCTTCTACCGGTCCCTGCTGGAAGATGAGGACATGGGCGAGCTGGTGGACGCCGAGGAATACCTGGTGCCTCAGCAGGGCTTCTTCTGCCCCGACCCTACCCCTGGCACCGGCTCTACCGCCCACAGACGGCACAGAAGCAGCAGCGCCAGAAACGGCGGAGGCGACCTGACCCTGGGAATGGAACCTAGCGGCGAGGGACCTCCCAGAAGCCCTAGAGCCCCTAGCGAGGGCACCGGCAGCGACGTGTTCGATGGCGATCTGGCCGTGGGCGTGACCAAGGGACTGCAGAGCCTGAGCCCCCAGGACCTGTCCCCCCTGCAGAGATACAGCGAGGACCCCACCCTGCCCCTGCCCAGCGAGACAGATGGCAAGGTGGCCCCCCTGAGCTGCAGCCCTCAGCCCGAGTTCGTGAACCAGAGCGACGTGCAGCCCAAGTCCCCCCTGACACCCGAGGGACCTCCAAGCCCTGCCAGACCTACCGGCGCCACCCTGGAAAGAGCCAAGACCCTGAGCCCCGGCAAGAACGGCGTGGTGAAAGACGTGTTCACCTTCGGAGGCGCCGTGGAAAACCCCGAGTTCCTGGCCCCCAGAGAGGGCACAGCCAGCCCTCCACACCCCAGCCCAGCCTTCTCCCCCGCCTTCGACAACCTGTTCTTCTGGGACCAGAACAGCAGCGAGCAGGGCCCACCCCCCAGCAATTTCGAGGGCACCCCCACCGCCGAGAATCCTGAGTTCCTGGGCCTGGACGTGCCCGTGTGA SEQ ID NO: 40. Amino acid sequence of heavychain of the anti-CD40 antibody CP870,893:MDWTWRILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. SEQ ID NO: 41. Acid sequence of the light chainof the anti-CD40 antibody CP870,893:MRLPAQLLGLLLLWFPGSRCDIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC. SEQ IDNO: 42. Acid sequence of the heavy chain of the anti-CTLA-4 antibodyTremelimumab QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDPRGATLYYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 43.Acid sequence of the light chain of the anti-CTLA-4 antibodyTremelimumab DIQMTQSPSSLSASVGDRVTITCRASQSINSYLDWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPFTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 44. Nucleotidesequence of CpG 7909 5′ TCGTCGTTTTGTCGTTTTGTCGTT3′ SEQ ID NO: 45.Nucleotide sequence of CpG 24555 5′ TCGTCGTTTTTCGGTGCTTTT3′ SEQ ID NO:46. Nucleotide sequence of CpG 10103 5′ TCGTCGTTTTTCGGTCGTTTT3′ SEQ IDNO: 47. Amino acid sequence of eGFPMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGI TLGMDELYK SEQID NO: 48. Amino acid sequence of HBV core antigenMDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGNNLEDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRGRSPRRRTPSPRRRRSQSP RRRRSQSRESQCSEQ ID NO: 49. Amino acid sequence of HBV surface antigenMENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSTTTSTGPCKTCTTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYI SEQ ID NO: 50. Aminoacid sequence of Rhesus PSMA ECD protein:MASETDTLLLWVLLLWVPGSTGDAAHHHHHHKSSSEATNITPKHNMKAFLDELKAENIKKFLHNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELTHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPAGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGATGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSASPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTSEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVYNLTKELESPDEGFEGKSLYESWTKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSSYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSVVLPFDCRDYAVVLRKYADKIYNISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLRDFDKSNPILLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSQAWGEVKRQISIATFTVQAAAETLSEVA SEQ ID NO: 51. Amino acidsequence of rat Her-2 p66 peptide (H-2d T cell epitope) TYVPANASL SEQ IDNO: 52. Amino acid sequence of rat Her-2 p169 peptide (H-2d T cellepitope) DMVLWKDVFRKNNQL SEQ ID NO: 53. Amino acid sequence of HBV coreantigen p87 peptide SYVNTNMGL SEQ ID NO: 54. Amino acid sequence of aRat Her-2 Antigen (rHer-2):MASELAAWCRWGFLLALLPPGIAGTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYVPANASLSFLQDIQEVQGYMLIAHNQVKRVPLQRLRIVRGTQLFEDKYALAVLDNRDPQDNVAASTPGRTPEGLRELQLRSLTEILKGGVLIRGNPQLCYQDMVLWKDVFRKNNQLAPVDIDTNRSRACPPCAPACKDNHCWGESPEDCQILTGTICTSGCARCKGRLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMHNPEGRYTFGASCVTTCPYNYLSTEVGSCTLVCPPNNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLRGARAITSDNVQEFDGCKKIFGSLAFLPESFDGDPSSGIAPLRPEQLQVFETLEEITGYLYISAWPDSLRDLSVFQNLRIIRGRILHDGAYSLTLQGLGIHSLGLRSLRELGSGLALIHRNAHLCFVHTVPWDQLFRNPHQALLHSGNRPEEDCGLEGLVCNSLCAHGHCWGPGPTQCVNCSHFLRGQECVEECRVWKGLPREYVSDKRCLPCHPECQPQNSSETCFGSEADQCAACAHYKDSSSCVARCPSGVKPDLSYMPIWKYPDEEGICQPCPINCTHSCVDLDERGCPAEQRASPVTFIIATVVGVLLFLILVVVVGILIKRRRQKIRKYTMRRNEDLGPSSPMDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFSPDPTPGTGSTAHRRHRSSSTRSGGGELTLGLEPSEEGPPRSPLAPSEGAGSDVFDGDLAMGVTKGLQSLSPHDLSPLQRYSEDPTLPLPPETDGYVAPLACSPQPEFVNQSEVQPQPPLTPEGPLPPVRPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEFLVPREGTASPPHPSPAFSPAFDNLFFWDQNSSEQGPPPSNFEGTPTAENPEFLGLDVPV SEQ ID NO: 55. Amino AcidSequence of Rhesus PSMA antigen:MASARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSSEATNITPKHNMKAFLDELKAENIKKFLHNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELTHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPAGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGATGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSASPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTSEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVYNLTKELESPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSSYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSVVLPFDCRDYAVVLRKYADKIYNISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLRDFDKSNPILLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSQAWGEVKRQISIATFTVQAAAETLSEVA SEQ ID NO: 56 Nucleotide sequence encoding therhesus PSMA antigen of SEQ ID NO: 55″ATGGCTAGCGCTAGAAGGCCCAGATGGCTGTGCGCTGGCGCCCTGGTGCTGGCTGGCGGATTCTTCCTGCTGGGCTTCCTGTTCGGCTGGTTCATCAAGTCCTCCAGCGAGGCCACCAACATCACCCCCAAGCACAACATGAAGGCCTTTCTGGACGAGCTGAAGGCCGAGAATATCAAGAAGTTCCTGCACAACTTCACCCAGATCCCCCACCTGGCCGGCACCGAGCAGAACTTCCAGCTGGCCAAGCAGATCCAGTCCCAGTGGAAAGAGTTCGGCCTGGACTCCGTGGAACTGACCCACTACGACGTGCTGCTGTCCTACCCCAACAAGACCCACCCCAACTACATCTCCATCATCAACGAGGACGGCAACGAAATCTTCAACACCTCCCTGTTCGAGCCCCCACCAGCCGGCTACGAGAACGTGTCCGACATCGTGCCCCCATTCTCCGCATTCAGTCCACAAGGCATGCCCGAGGGCGACCTGGTGTACGTGAACTACGCCAGGACCGAGGACTTCTTCAAGCTGGAAAGGGACATGAAGATCAACTGCTCCGGCAAGATCGTGATCGCCAGATACGGCAAGGTGTTCAGGGGCAACAAAGTGAAGAACGCTCAGCTGGCTGGGGCCACCGGCGTGATCCTGTACTCTGACCCCGCCGACTACTTCGCCCCAGGCGTGAAGTCCTACCCCGACGGCTGGAACCTGCCAGGTGGCGGAGTGCAGAGGGGCAACATCCTGAACCTGAACGGCGCTGGCGACCCCCTGACCCCAGGATACCCCGCCAACGAGTACGCCTACAGAAGAGGAATCGCCGAGGCCGTGGGCCTGCCCTCTATCCCAGTGCACCCCATCGGCTACTACGACGCCCAGAAACTGCTGGAAAAGATGGGCGGCTCCGCCTCCCCCGACTCCTCTTGGAGAGGCTCCCTGAAGGTGCCCTACAACGTGGGCCCAGGCTTCACCGGCAACTTCTCCACCCAGAAAGTGAAGATGCACATCCACTCCACCTCCGAAGTGACCAGGATCTACAACGTGATCGGCACCCTGAGAGGCGCCGTGGAACCCGACAGATACGTGATCCTGGGCGGCCACAGGGACAGCTGGGTGTTCGGCGGCATCGACCCACAGTCTGGCGCCGCTGTGGTGCACGAGATCGTGCGGTCCTTCGGAACCCTGAAGAAAGAGGGATGGCGCCCCAGAAGGACAATCCTGTTCGCCTCCTGGGACGCCGAGGAATTCGGCCTGCTGGGATCCACCGAGTGGGCCGAGGAAAACTCCAGGCTGCTGCAGGAAAGGGGCGTCGCCTACATCAACGCCGACTCCTCCATCGAGGGCAACTACACCCTGAGGGTGGACTGCACCCCCCTGATGTACTCCCTGGTGTACAACCTGACCAAAGAGCTGGAATCCCCCGACGAGGGCTTCGAGGGCAAGTCCCTGTACGAGTCCTGGACCAAGAAGTCCCCATCCCCCGAGTTCTCCGGCATGCCCAGGATCTCCAAGCTGGGCTCCGGCAACGACTTCGAGGTGTTCTTCCAGAGGCTGGGAATCGCCTCCGGCAGGGCCAGATACACCAAGAACTGGGAGACAAACAAGTTCTCCTCCTACCCCCTGTACCACTCCGTGTACGAAACCTACGAGCTGGTGGAAAAGTTCTACGACCCCATGTTCAAGTACCACCTGACCGTGGCCCAGGTCCGCGGAGGCATGGTGTTCGAGCTGGCCAACTCCGTGGTGCTGCCCTTCGACTGCAGAGACTATGCTGTGGTGCTGAGGAAGTACGCCGACAAAATCTACAACATCTCCATGAAGCACCCCCAGGAAATGAAGACCTACTCCGTGTCCTTCGACTCCCTGTTCTCCGCCGTGAAGAATTTCACCGAGATCGCCTCCAAGTTCTCCGAGAGGCTGAGGGACTTCGACAAGTCCAACCCCATCCTGCTGAGGATGATGAACGACCAGCTGATGTTCCTGGAAAGGGCCTTCATCGACCCCCTGGGCCTGCCAGACAGGCCCTTCTACAGGCACGTGATCTACGCCCCATCCTCCCACAACAAATACGCCGGCGAGTCCTTCCCCGGCATCTACGATGCCCTGTTCGACATCGAGTCCAAGGTGGACCCCTCCCAGGCTTGGGGCGAAGTGAAGAGGCAGATCAGTATCGCCACATTCACAGTGCAGGCCGCTGCCGAAACCCTGTCCGAGGTGGCC

1-5. (canceled)
 6. A method of treating prostate cancer in a human,comprising administering to the human an effective amount of acomposition 1 comprising a multi-antigen construct, wherein themulti-antigen construct comprises: (a) at least one nucleotide sequenceencoding an immunogenic PSA polypeptide; (b) at least one nucleotidesequence encoding an immunogenic PSCA polypeptide; and (c) at least onenucleotide sequence encoding an immunogenic PSMA polypeptide, whereinthe immunogenic PSA polypeptide comprises amino acids 4-240 of SEQ IDNO:17, wherein the immunogenic PSCA polypeptide comprises the amino acidsequence of SEQ ID NO:21, and wherein the immunogenic PSMA polypeptidehas at least 90% identity with amino acids 15-750 of the human PSMA ofSEQ ID NO:1 and comprises the amino acids of at least 10 conserved Tcell epitopes of the human PSMA at corresponding positions. 7-26.(canceled)
 27. The method according claim 6, wherein the immunogenicPSMA polypeptide is selected from the group consisting of: 1) apolypeptide comprising amino acids 15-750 of SEQ ID NO: 1; 2) apolypeptide comprising the amino acid sequence of SEQ ID NO:3; 3) apolypeptide comprising the amino acid sequence of SEQ ID NO:5; 4) apolypeptide comprising the amino acid sequence of SEQ ID NO:7; 5) apolypeptide comprising the amino acids 4-739 of SEQ ID NO:9; 6) apolypeptide comprising the amino acids 4-739 of SEQ ID NO:3; 7) apolypeptide comprising the amino acids 4-739 of SEQ ID NO:5; 8) apolypeptide comprising the amino acids 4-739 of SEQ ID NO:7; and 9)polypeptide comprising the amino acid sequence of SEQ ID NO:
 9. 28. Themethod according to claim 27, wherein the nucleotide sequence encodingthe immunogenic PSA polypeptide is set forth in SEQ ID NO:18.
 29. Themethod according to claim 28, wherein the nucleotide sequence encodingthe immunogenic PSMA polypeptide is selected from the group consistingof: 1) the nucleotide sequence of SEQ ID NO:2; 2) the nucleotidesequence of SEQ ID NO:4; 3) the nucleotide sequence of SEQ ID NO:6; 4)the nucleotide sequence of SEQ ID NO:8; 5) the nucleotide sequence ofSEQ ID NO:10; 6) a nucleotide sequence comprising nucleotides 10-2217 ofSEQ ID NO:4; 7) a nucleotide sequence comprising nucleotides 10-2217 ofSEQ ID NO:6; 8) a nucleotide sequence comprising nucleotides 10-2217 ofSEQ ID NO:8; and 9) a nucleotide sequence comprising nucleotides 10-2217of SEQ ID NO:10.
 30. The method according to claim 6, wherein themulti-antigen construct is incorporated into a vector.
 31. The methodaccording to claim 30, wherein the multi-antigen construct furthercomprises: (a) a nucleotide sequence encoding a T2A peptide sequence;and (b) a nucleotide sequence encoding a F2A peptide sequence.
 32. Themethod according to 31, wherein the order of the nucleotide sequences onthe multi-antigen construct is shown in formula (I):PSA-T2A-PSCA-F2A-PSMA (I) wherein in formula (I): PSA is the nucleotidesequence encoding the immunogenic PSA polypeptide; PSCA is thenucleotide sequence encoding the immunogenic PSCA polypeptide; PSMA isthe nucleotide sequence encoding the immunogenic PSMA polypeptide; T2Ais the nucleotide sequence encoding the T2A peptide sequence; and F2A isthe nucleotide sequence encoding the F2A peptide sequence.
 33. Themethod according to 32, wherein the immunogenic PSMA polypeptidecomprises the amino acid sequence of SEQ ID NO:
 9. 34. The methodaccording to claim 32, wherein the nucleotide sequence encoding theimmunogenic PSMA polypeptide is set forth in SEQ ID NO:10.
 35. A methodof treating prostate cancer in a human, comprising administering to thehuman an effective amount of a composition comprising multi-antigenconstruct, wherein the multi-antigen construct comprises the nucleotidesequence of SEQ ID NO:35 or a degenerate variant of the nucleotidesequence of SEQ ID NO:35.
 36. The method according to claim 35, whereinthe multi-antigen construct comprises a degenerate variant of thenucleotide sequence of SEQ ID NO:35.
 37. The method according to claim35, further comprising administering to the human an effective amount ofan immune modulators.
 38. The method according to claim 37, wherein theimmune modulator is an immune-effector-cell enhancer.
 39. The methodaccording to claim 38, wherein the immune-effector-cell enhancer isselected from the group consisting of TNFR agonists, CTLA-4 antagonists,TLR agonists, programmed cell death protein 1 antagonists, programmedcell death protein 1 ligand antagonists.
 40. The method according toclaim 39, wherein the immune-effector-cell enhancer is a CTLA-4antagonist.
 41. The method according to claim 40, wherein the CTLA-4antagonist is an anti-CTLA-4 antibody.
 42. The method according to claim39, wherein the immune-effector-cell enhancer is a TLR agonist.
 43. Themethod according to claim 42, wherein the TLR agonist is a CpGoligonucleotide.
 44. The method according to claim 37, wherein theimmune modulator is an immune-suppressive-cell inhibitor.
 45. The methodaccording to claim 44, wherein the immune-suppressive-cell inhibitor issunitinib malate.